Human mtDNA Genome Guide (15037958 01)

Human mtDNA Genome Guide (15037958 01)
Human mtDNA Genome
For the Illumina Sequencing Platform
IMPORTANT NOTICE
This document provides information for an application for Illumina technology that has been
demonstrated internally and may be of interest to customers. This information is provided as‐is and is
not an Illumina product and is not accompanied by any rights or warranties. Customers using or
adapting this information should obtain any licenses required and materials from authorized vendors.
Illumina products mentioned herein are for research use only unless marked otherwise, and are subject
to any and all applicable terms and conditions of use. While customer feedback is welcomed, not all
components of this application are supported by Illumina Technical Support and Field Application
Scientists.
Revision History
Introduction
Workflow
Perform First Amplification
Quantify DNA
Tagment Input DNA
Perform Second Amplification
Perform Second DNA Clean Up
Bead‐based Normalization
Pool Bead‐Based Normalized Libraries
Bioanalyzer‐based Normalization
Pool Bioanalyzer Normalized Libraries
Next Steps for Sequencing and Analysis
mtDNA Variant Processor BaseSpace App
mtDNA Variant Analyzer BaseSpace App
MiSeq Reporter
Supporting Information
Document # 15037958 v01
1
3
5
6
8
10
12
15
17
19
20
22
23
24
33
41
50
Page 1
Revision History
Page 1
Revision History
Document
Date
Document # 15037958
v01
February
2016
Description of Change
Clarified analysis requirements.
Updated incubation times for PCR clean up.
Updated PCR conditions.
Clarified analysis requirements.
Revised legal notice.
Added directions to transfer supernatent from CAA to CAN
plate in the second DNA cleanup.
Added a safe stopping point after quantification and the first
PCR amplification.
Updated the Edit Manifest section.
Corrected the number of cycles needed for sequencing a
library from 2 x 151 to 2 x 251.
Removed the following sections:
• Dual Indexing Principle
• Low Plexity Pooling Guidelines
Added directions for using the the BaseSpace mtDNA
Processor app and the BaseSpace mtDNA Variant Analyizer
app.
Updated design of workflow diagram.
Renamed and combined some procedures as needed to
improve continuity.
Simplified consumables information at the beginning of each
section.
Added tips and techniques section.
Revised step‐by‐step instructions to be more succinct.
For final transfer of Perform Second DNA Clean Up, replaced
midi plate labeled LNP with TCY plate labeled CAN.
For Bead‐based Normalization:
• Added midi plate to user‐supplied consumables
• Added instructions to label midi plate LNP
• Added initial transfer step from CAA plate to CAN plate
Added the following to the consumables and equipment list: • MiSeq FGx
• MiSeq Reagent Kit v3
• MiSeq FGx Reagent Kit v3
• Nextera XT DNA Library Prep v2 kits
• Optional microplate shakers
Corrected MiSeq Reagent Kit v2 catalog number.
Removed dilute and denature procedures. See the MiSeq
System Denature and Dilute Libraries Guide (document #
15039740) and follow the instructions for bead‐based
normalization and Nextera XT.
Revision History
Page 2
Document
Date
15037958 Rev. B
October
2013
15037958 Rev. A
September
2013
Description of Change
Added safe stopping point at the end of the Limited‐Cycle
PCR Clean‐Up procedure.
Added Nextera XT DNA Sample Preparation Kit (96 samples)
and Nextera XT DNA Sample Preparation Index Kits to the
consumables list.
Initial release.
Introduction
Page 3
Introduction
This protocol explains how to prepare, sequence, and analyze the entire human
mitochondrial DNA (mtDNA) genome from clean, intact DNA samples. During sample
preparation, the mtDNA genome is amplified in two PCRs to generate two long fragments
spanning the entire human mitochondrial genome (16,569 bp). The amplicons are quantified
and pooled before library preparation. Subsequent library sequencing on the MiSeq is
followed by data analysis.
NOTE
The protocols described in this guide assume that you have reviewed the Supporting
Information, on page 50 and obtained all of the requisite consumables and equipment.
Tips and Techniques
Unless a safe stopping point is specified in the protocol, proceed immediately to the next
step.
Avoiding Cross‐Contamination
• When adding or transferring samples, change tips between each sample.
• When adding adapters or primers, change tips between each row and each column.
• Remove unused index adapter tubes from the working area.
Sealing the Plate
• Always seal the 96‐well plate before the following steps in the protocol:
• Shaking steps
• Vortexing steps
• Centrifuge steps
• Thermal cycling steps
• Apply the adhesive seal to cover the plate and seal with a rubber roller.
• Microseal 'B' adhesive seals are effective at ‐40°C to 110°C, and suitable for skirted or
semiskirted PCR plates. Use Microseal 'B' for shaking, centrifuging, and long‐term
storage.
• Microseal 'A' adhesive film is effective for thermal cycling and easy to cut when using
fewer than 96 wells.
Plate Transfers
• When transferring volumes between plates, transfer the specified volume from each well
of a plate to the corresponding well of the other plate.
Centrifugation
• Centrifuge at any step in the procedure to consolidate liquid or beads in the bottom of
the well, and to prevent sample loss.
Handling Beads
• Pipette bead suspension slowly.
Introduction
Page 4
• When mixing, mix thoroughly.
• If beads are aspirated into the pipette tips, dispense back to the plate on the magnetic
stand and wait until the liquid is clear (~2 minutes).
• When washing beads:
• Use the appropriate magnet for the plate.
• Dispense liquid so that beads on the side of the wells are wetted.
• Keep the plate on the magnet until the instructions specify to remove it.
• Do not agitate the plate while on the magnetic stand. Do not disturb the bead pellet.
Prepare for Pooling
If you plan to pool libraries, record information about your samples before beginning library
prep. Different methods are available depending on the sequencing instrument you are
using. See the Human mtDNA Genome Sample Preparation support page for more
information.
Workflow
Page 5
Workflow
The following figure illustrates the Human mtDNA Genome workflow.
Figure 1
Human mtDNA Genome Workflow (for 12 reactions, 1‐200 pg/µl in 10 µl total DNA)
Perform First Amplification
Page 6
Perform First Amplification
This process generates two long PCR amplicons (9.1 kb and 11.2 kb) spanning the entire
human mtDNA genome. This procedure is for 12 samples (24 reactions).
Consumables
•
•
•
•
•
•
•
•
•
2.5 mM dNTP Mix (16 µl per sample)
10X LA PCR Buffer II (10 µl per sample)
96‐well 0.3 ml PCR plate
200 µl PCR tubes (2)
Microseal ‘B’ Adhesive Seal
Nuclease‐free water (45 µl per sample + volume to dilute primers)
PCR Primers (MTL‐F1, MTL‐R1, MTL‐F2, MTL‐R2) (1 tube of each primer)
Purified Human DNA (100 pg/µl)
TaKaRa LA TaqDNA Polymerase (1µl per sample)
Preparation
1
Prepare the following consumables.
Item
Storage
Instructions
2.5 mM dNTP Mix
‐25°C to ‐15°C
Thaw at room
temperature.
10X LA PCR Buffer II
‐25°C to ‐15°C
Thaw at room
temperature.
PCR Primers
(MTL‐F1, MTL‐R1, MTL‐F2, MTL‐R2)
‐25°C to ‐15°C
Thaw at room
temperature.
Purified Human DNA
‐25°C to ‐15°C
Thaw at room
temperature.
TaKaRa LA TaqDNA Polymerase
‐25°C to ‐15°C
Remove from
storage. Keep on ice.
2
Pre‐program the thermal cycler with the following program and save as LA PCR:
• Choose the pre‐heat lid option and set to 100°C
• 94°C for 5 minutes
• 30 cycles of:
• 98°C for 15 seconds
• 68°C for 10 seconds (slow ramp from 68°C to 60°C at 0.2°C per second)
• 60°C for 15 seconds
• 68°C for 11 minutes
• 72°C for 10 minutes
• Hold at 10°C
Perform First Amplification
Page 7
3
Use a pen to label 2 new 200 µl PCR tubes as MTL1 and MTL2.
Procedure
1
Dilute each PCR primer to 10 µM with nuclease‐free water.
Store excess primer for future use at ‐15C° to ‐25°C.
2
For each primer pair (MTL‐F1 + MTL‐R1; MTL‐F2 + MTL‐R2), prepare the following PCR
master mix in the 200 µl PCR tube labeled with the appropriate primer pair number
(MTL1 or MTL2). Pipette to mx.
The reaction master mix can be scaled up for multiple samples.
NOTE
Do not let this PCR master mix stand. Following mixing, proceed immediately to step 3.
Reagent
10X LA PCR Buffer II (25 mM MgCl2 )
Diluted Forward Primer (e.g., MTL‐F1 or MTL‐F2)
Diluted Reverse Primer (e.g., MTL‐R1 or MTL‐R2)
2.5 mM dNTP mix
Nuclease‐free water
TaKaRa LA Taq (5 U/µl)
Total
Volume (µl)
5
2
2
8
22.5
0.5
40
3
Add 40 µl PCR master mix from step 2 to each of two separate, sterile, nuclease‐free,
200 µl PCR tubes or each well of a new 96‐well 0.3 ml PCR plate, one tube or well of
each primer master mix per sample.
4
Add 10 µl purified Human DNA to each plate well, then pipette to mix.
5
Place the tube or plate on the pre‐programmed thermal cycler. Close the lid and select
LA PCR to amplify the plate.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at ‐25°C to ‐15°C for up to 7 days.
Quantify DNA
Page 8
Quantify DNA
This step determines the quantity of the mitochondrial amplicons (MTL‐F1 + MTL‐R1 and
MTL‐F2 + MTL‐R2) available for library preparation.
NOTE
Perform this and all subsequent procedures in a post‐PCR lab.
Consumables
•
•
•
•
RSB (Resuspension Buffer)
2 ml microcentrifuge tubes (2)
96‐well 0.3 ml PCR plate
Agilent DNA 12000 Kit
Procedure
1
Verify the size distribution of each PCR amplicon by running a 1 µl aliquot on Agilent
DNA 12000 chip using an Agilent Technologies 2100 Bioanalyzer.
NOTE
These fragments are close to the larger marker and are not accurate. The size range should be
7000–9000 bp.
2
Primer pairs MTL‐F1 + MTL‐R1 should generate 9065 bp. This should appear as a single
peak in the range of 7705–10425 bp.
Figure 2
Example of Electropherogram of the MTL‐F1 + MTL‐R1 PCR Amplicon
Quantify DNA
Page 9
3
Primer pairs MTL‐F2 + MTL‐R2 should generate 11170 bp. This should appear as a
single peak in the range of 9495–12845 bp.
Figure 3
Example of Electropherogram of the MTL‐F2 + MTL‐R2 PCR Amplicon
4
Determine the concentration of the PCR amplicon by integrating the area under the peak
following manufacturer’s instructions.
5
Normalize each PCR amplicon with RSB in a separate microcentrifuge tube to a
concentration of 0.2 ng/µl. Pipette to mix.
6
Transfer an equal volume of 10 µl each PCR amplicon to 1 well of a new 0.3 ml PCR
plate for each sample. Pipette to mix.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at ‐25°C to ‐15°C for up to 16 hours.
Tagment Input DNA
Page 10
Tagment Input DNA
This step uses the Human mtDNA Genome Sample Prep transposome to tagment input
DNA, which is a process that fragments DNA and then tags the DNA with an adapter
sequence in a single step.
Consumables
•
•
•
•
•
•
•
ATM (Amplicon Tagment Mix)
TD (Tagment DNA Buffer)
NT (Neutralize Tagment Buffer)
96‐well hard shell TCY plate
Input DNA (0.2 ng/µl)
Microseal 'B' adhesive seals (2)
PCR 8‐tube strip with caps (3)
Preparation
1
Prepare the following consumables:
Item
Input
DNA
ATM
TD
NT
Storage
Instructions
‐25°C to ‐15°C Thaw on ice. Gently invert the thawed tubes 3–5 times, and then
centrifuge briefly.
‐25°C to ‐15°C Thaw on ice. Gently invert the thawed tubes 3–5 times, and then
centrifuge briefly.
‐25°C to ‐15°C Thaw on ice. Gently invert the thawed tubes 3–5 times, and then
centrifuge briefly.
15°C to 30°C Check for precipitates. If present, vortex until all particulates are
resuspended.
2
Save the following tagmentation program on the thermal cycler:
• Choose the preheat lid option
• 55°C for 5 minutes
• Hold at 10°C
3
Calculate the total volume of ATM for all reactions and divide the volume equally
among the wells of a PCR 8‐tube strip.
4
Use a pen to label a new 96‐well TCY plate NTA.
Procedure
1
Add the following items in the order listed to each well of the NTA plate. Pipette to mix.
Item
TD
gDNA (1 ng total)
2
Add 5 µl ATM to each well. Pipette to mix.
3
Centrifuge at 280 × g at 20°C for 1 minute.
Volume (µl)
10
5
Tagment Input DNA
Page 11
4
Place on the programmed thermal cycler and run the tagmentation program.
5
When the sample reached 10°C, immediately add 5 µl NT to each well. Pipette to mix.
6
Centrifuge at 280 × g at 20°C for 1 minute.
7
Incubate at room temperature for 5 minutes.
Perform Second Amplification
Page 12
Perform Second Amplification
This step amplifies the tagmented DNA using a limited‐cycle PCR program. The PCR step
also adds index 1 (i7) adapters and index 2 (i5) adapters and sequences required for cluster
formation. Use the full amount of recommended input DNA. To ensure libraries produce
high‐quality sequencing results, use the specified number of PCR cycles.
Consumables
•
•
•
•
•
NPM (Nextera PCR Master Mix)
Index 1 primers (N7XX)
Index 2 primers (S5XX)
TruSeq Index Plate Fixture
Microseal 'A' film
Preparation
1
Prepare the following consumables.
Item
Index adapters (i5 and i7)
NPM
Storage
‐25°C to ‐15°C
‐25°C to ‐15°C
Instructions
Thaw at room temperature for 20 minutes.
Thaw at room temperature for 20 minutes.
2
Save the following program on a thermal cycler:
• 72°C for 3 minutes
• 95°C for 30 seconds
• 12 cycles of:
• 95°C for 1 seconds
• 55°C for 30 seconds
• 72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 10°C
3
Calculate the total volume of NPM for all reactions and divide the volume equally
among the wells of a PCR 8‐tube strip.
Procedure
1
[24 libraries] Arrange the index primers in the TruSeq Index Plate Fixture as follows.
• Arrange Index 1 (i7) adapters in columns 1–6 of the TruSeq Index Plate Fixture.
• Arrange Index 2 (i5) adapter in rows A–D of the TruSeq Index Plate Fixture.
Perform Second Amplification
Page 13
Figure 4
TruSeq Index Plate Fixture Arrangement (24 libraries)
A Rows A–D: Index 2 (i5) adapters (white caps)
B Columns 1–6: Index 1 (i7) adapters (orange caps)
C NTA plate
2
[96 libraries] Arrange the index primers in the TruSeq Index Plate Fixture as follows.
• Arrange Index 1 (i7) adapters in columns 1–12 of the TruSeq Index Plate Fixture.
• Arrange Index 2 (i5) adapter in rows A–H of the TruSeq Index Plate Fixture.
Figure 5
TruSeq Index Plate Fixture (96 libraries)
D Rows A–H: Index 2 (i5) adapters (white caps)
E Columns 1–12: Index 1 (i7) adapters (orange caps)
F NTA plate
Perform Second Amplification
Page 14
3
Use a multichannel pipette to add 5 µl of each Index 1 (i7) adapter down each column.
Replace the cap on each i7 adapter tube with a new orange cap.
4
Use a multichannel pipette to add 5 µl of each Index 2 (i5) adapter across each row.
Replace the cap on each i5 adapter tube with a new white cap.
5
Add 15 µl NPM to each well containing index adapters. Pipette to mix.
6
Centrifuge at 280 × g at 20°C for 1 minute.
7
Place on the preprogrammed thermal cycler and run the PCR program.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at 2°C to 8°C for up to 2 days. Alternatively,
leave on the thermal cycler overnight.
Perform Second DNA Clean Up
Page 15
Perform Second DNA Clean Up
This step uses AMPure XP beads to purify the library DNA and provides a size selection
step that removes short library fragments.
Consumables
•
•
•
•
•
•
RSB (Resuspension Buffer)
AMPure XP beads
Freshly prepared 80% ethanol (EtOH)
96‐well midi plate
96‐well TCY plate
Nuclease‐free water
About Reagents
• Vortex AMPure XP beads before each use.
• Vortex AMPure XP beads frequently to make sure that beads are evenly distributed.
• Always prepare fresh 80% ethanol for wash steps. Ethanol can absorb water from the air
impacting your results.
Preparation
1
Prepare the following consumables:
Item
Storage
Instructions
RSB
‐25°C to ‐15°C Thaw at room temperature.
AMPure XP Beads 2°C to 8°C
Let stand for 30 minutes to bring to room temperature.
2
Prepare fresh 80% ethanol from absolute ethanol.
3
Use a pen to label a new midi plate CAA (Clean Amplified Plate).
4
Use a pen to label a new TCY plate CAN (Clean Amplified NTA Plate).
Procedure
1
Centrifuge at 280 × g at 20°C for 1 minute.
2
Transfer 50 µl PCR product from each well of the NTA plate to corresponding wells of
the CAA plate.
3
Add 30 µl AMPure XP beads to each well of the midi plate.
4
Pipette to mix or shake at 1800 rpm for 2 minutes.
5
Incubate at room temperature for 5 minutes.
6
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
7
Remove and discard all supernatant from each well.
Perform Second DNA Clean Up
Page 16
8
Wash 2 times as follows.
a Add 200 µl fresh 80% EtOH to each well.
b Incubate on the magnetic stand for 30 seconds.
c Remove and discard all supernatant from each well.
9
Using a 20 µl pipette, remove residual 80% EtOH from each well.
10 Air‐dry on the magnetic stand for 15 minutes.
11 Remove from the magnetic stand.
12 Add 52.5 µl RSB to each well.
13 Pipette to mix or shake at 1800 rpm for 2 minutes.
14 Incubate at room temperature for 2 minutes.
15 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
16 Transfer 50 µl supernatant from the CAA plate to the CAN plate.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at ‐25°C to ‐15°C for up to 7 days.
Bead-based Normalization
Page 17
Bead‐based Normalization
This step uses beads to normalize the quantity of each library, which ensures more equal
library representation in your pooled sample.
NOTE
Manually normalize libraries when the final library yield is less than 10–15 nM. Bead‐based
normalization on low yield libraries can result in overly diluted samples and low sequencing
yields. See Manually Normalizing Libraries: Best Practices on MyIllumina.com.
Consumables
•
•
•
•
•
•
•
•
•
LNA1 (Library Normalization Additives 1)
LNB1 (Library Normalization Beads 1)
LNW1 (Library Normalization Wash 1) (2 tubes)
LNS1 (Library Normalization Storage Buffer 1)
0.1 N NaOH (fewer than 7 days old) (3 ml per 96 samples)
96‐well midi plate
96‐well TCY plate
15 ml conical tube
Microseal 'B' adhesive seals (5)
About Reagents
• Vortex LNA1 vigorously to make sure that all precipitates have dissolved. Inspect in
front of a light.
• Vortex LNB1 vigorously, with intermittent inversion (at least 1 minute). Repeat until all
beads are resuspended and no beads are present at the bottom of the tube when it is
inverted.
• Use only the P1000 pipette set to 1000 µl even if you are using less than full tubes.
Never use a P200 pipette to handle LNB1. Mix only the required amounts of LNA1 and
LNB1 for the current experiment. Store the remaining LNA1 and LNB1 separately at
their recommended temperatures.
• Aspirate and dispense beads slowly due to the viscosity of the solution.
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact,
and eye contact. Wear protective equipment, including eye protection, gloves, and
laboratory coat. Handle used reagents as chemical waste and discard in accordance with
the governmental safety standards for your region. For environmental, health, and safety
information, see the SDS for this kit at support.illumina.com/sds.html.
Preparation
1
Prepare the following consumables:
Bead-based Normalization
Page 18
Item Storage
LNA1 ‐25°C to ‐15°C
Instructions
Prepare under a fume hood.
Bring to room temperature. Use a 20°C to 25°C water bath as needed.
LNB1 2°C to 8°C
Bring to room temperature. Use a 20°C to 25°C water bath as needed.
LNW1 2°C to 8°C
Bring to room temperature. Use a 20°C to 25°C water bath as needed.
LNS1 Room temperature Bring to room temperature.
2
Label a new 96‐well TCY plate as SGP (Storage Plate).
3
Label a new 96‐well midi plate as LNP (Library Normalization Plate).
Procedure
1
Transfer 20 µl supernatant from the TCY plate to a new midi plate.
2
[96 samples] Add 4.4 ml LNA1 to a new 15 ml conical tube.
3
Thoroughly resuspend LNB1. Pipette to mix.
4
Transfer 800 µl LNB1 to the 15 ml conical tube containing LNA1. Invert to mix.
5
Pour the bead mixture into a trough and add 45 µl combined LNA1/LNB1 to each well
containing libraries.
6
Shake at 1800 rpm for 30 minutes.
7
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
8
Remove and discard all supernatant from each well.
9
Wash 2 times as follows.
a
b
c
d
Add 45 µl LNW1 to each well.
Shake at 1800 rpm for 5 minutes.
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
Remove and discard all supernatant from each well.
10 Add 30 µl 0.1 N NaOH to each well.
11 Shake at 1800 rpm for 5 minutes.
12 Add 30 µl LNS1 to each well of the SGP plate. Set aside.
13 After the 5 minute elution, make sure that all samples in the midi plate are resuspended.
Pipette to mix or lightly tap the plate on the bench.
14 Shake at 1800 rpm for 5 minutes.
15 Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
16 Transfer the supernatant from the midi plate to the SGP plate.
17 Centrifuge at 1000 × g for 1 minute.
NOTE
Use qPCR for quality control, if desired. For more information, see the Sequencing Library
qPCR Quantification Guide (part # 11322363).
SAFE STOPPING POINT
If you are stopping, seal the plate and store at ‐25°C to ‐15°C for up to 7 days.
Pool Bead-Based Normalized Libraries
Page 19
Pool Bead‐Based Normalized Libraries
This step combines equal volumes of bead‐based normalized libraries in a single tube. After
pooling, dilute and heat‐denature the library pool before loading libraries for the sequencing
run.
Consumables
• MiSeq reagent cartridge (MiSeq Reagent Kit contents)
• Eppendorf tubes with screw caps (2)
• PCR 8‐tube strip
Preparation
1
To prepare for the sequencing run, begin thawing the reagent cartridge according to the
instructions for your MiSeq sequencing instrument.
2
If the SGP plate was stored frozen at ‐25°C to ‐15°C, thaw at room temperature. Pipette to
mix.
Procedure
1
Centrifuge SGP plate at 1000 × g at 20°C for 1 minute.
2
Transfer 5 µl of each library from the SGP plate to a new PCR 8‐tube strip.
3
Use a pen to label a new Eppendorf tube PAL.
4
Transfer the contents of the PCR 8‐tube strip to the PAL tube. Invert to mix.
5
Dilute pooled libraries to the loading concentration. See the MiSeq System Denature and
Dilute Libraries Guide (document # 15039740) and follow the instructions for bead‐based
normalization and Nextera XT.
6
Store unused pooled libraries in the PAL tube and PCR plate at ‐25°C to ‐15°C for up to 7
days.
7
Sequence your library using a 2 x 251‐cycle run with 2 index reads on the MiSeq system,
and then perform analysis. See Next Steps for Sequencing and Analysis, on page 23.
Bioanalyzer-based Normalization
Page 20
Bioanalyzer‐based Normalization
As an alternative to bead‐based normalization, this step uses a bioanalyzer to determine the
molarities from the sample region for normalization of the library.
Consumables
• Agilent High Sensitivity DNA Kit
• PCR 8‐tube strip
• RSB (Resuspension Buffer)
Preparation
1
Prepare the following consumables:
Item
RSB
Agilent High Sensitivity DNA Kit
Storage
‐25°C to ‐15°C
2°C to 8°C
Instructions
Thaw at room temperature.
Bring to room temperature.
Procedure
1
Analyze 1 µl of each PCR on the Bioanalyzer with a High Sensitivity DNA Chip by
following manufacturer instructions.
Figure 6
Example Library
2
Select the desired sample trace to display the electropherogram on the right side of
window.
3
Select the Region Table tab.
4
Right‐click the electropherogram, and then select Add Region.
Bioanalyzer-based Normalization
Page 21
5
Click and drag the blue region lines to capture the entire DNA library range as shown.
Molarity appears in the table below the electropherogram. If it does not appear, right‐
click the table, and click the configure columns. Make sure that molarity appears in the
displayed column.
Figure 7
Example Region Of Interest
6
Normalize each DNA sample library to 2 nM with RSB in a separate well of an 8‐tube
strip.
7
Proceed to Pool and Load from Bioanalyzer-based Normalization on page 1.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at ‐25°C to ‐15°C for up to 3 days.
Pool Bioanalyzer Normalized Libraries
Page 22
Pool Bioanalyzer Normalized Libraries
If you normalized your libraries with a bioanalyzer, this step combines equal volumes of
Bioanalyzer normalized libraries in a single tube. After pooling, dilute and heat‐denature the
library pool before loading libraries for the sequencing run.
Consumables
•
•
•
•
•
•
HT1 (Hybridization buffer) (Nextera XT DNA Library Prep Kit contents)
MiSeq reagent cartridge (MiSeq Reagent Kit contents)
Freshly diluted 0.2 N NaOH (5 µl)
Microcentrifuge tubes (3)
PhiX Control v3
Tris‐HCl, pH 8.5 with 0.1% Tween 20
Preparation
1
Remove HT1 and MiSeq reagent cartridge from ‐25°C to ‐15°C storage and thaw at room
temperature.
Procedure
1
Add 5 µl of each 2 nM normalized library to 1 microcentrifuge tube.
2
Dilute pooled libraries to the loading concentration. See the MiSeq System Denature and
Dilute Libraries Guide (document # 15039740).
3
Sequence your library using a 2 x 251‐cycle run with 2 index reads on the MiSeq system.
See Next Steps for Sequencing and Analysis, on page 23.
Next Steps for Sequencing and Analysis
Page 23
Next Steps for Sequencing and Analysis
After the denaturation and dilution of DNA, load sample libraries into the MiSeq reagent
cartridge and perform a sequencing run on the MiSeq or MiSeq FGx instrument. See the
guide for your MiSeq instrument for complete information.
BaseSpace software is recommended for analysis. If you use BaseSpace, select the BaseSpace
option when setting up the sequencing run on the MiSeq.
In BaseSpace, the BaseSpace mtDNA Variant Processor App performs the alignment and
variant calling of samples. The BaseSpace mtDNA Variant Analyzer App then interrogates
the results from the variant processor to analyze mitochondrial DNA. See mtDNA Variant
Processor BaseSpace App, on page 24 and mtDNA Variant Analyzer BaseSpace App, on
page 33.
As an alternative, analysis of samples from the sequencing run can be performed using the
mtDNA MiSeq Reporter Plug‐In with a compatible version of the MiSeq Reporter software
and the mtDNA Variant Analyzer application. Note that the mtDNA Variant Analyzer is
not the same as the BaseSpace mtDNA Variant Analyzer App. See MiSeq Reporter, on page
41.
mtDNA Variant Processor BaseSpace App
Page 24
mtDNA Variant Processor BaseSpace App
The BaseSpace® Variant Processor BaseSpace App performs alignment and variant calling of
samples against a reference mitochondrial DNA (mtDNA) genome. The app can analyze any
part of the full circular genome using quality and coverage thresholds information. The app
produces BAM and VCF output files. You can use the BAM files for alignment research with
third party tools. You can view the analysis results of the VCF files in the mtDNA Variant
Analyzer App.
See the mtDNA Variant Processor App Guide (document# 1000000007931) for complete
information on the application.
Compatible Libraries
See the BaseSpace support page for a list of library types that are compatible with the
Analysis App.
Versions
The following components are used in the Analysis App.
Software
Version
Isas (Analysis Software)
2.6.7.4
BWA-MEM (Aligner)
0.7.9
Workflow Requirements
• Supports up to 96 samples per alignment.
• Requires alignment to the Revised Cambridge Reference Sequence (rCRS)
• Supports data from samples prepared with the Human mtDNA D‐Loop Hypervariable
Region Guide (document# 15034858) and Human mtDNA Genome Region Guide
(document# 15037958).
Reference Genomes
The genome for alignment is the Revised Cambridge Reference Sequence (rCRS), a mtDNA
genome used by the Forensic Genomics community.
mtDNA Variant Processor BaseSpace App
Page 25
Workflow Diagram
Figure 8
mtDNA Variant Processor Workflow
mtDNA Variant Processor BaseSpace App
Page 26
Workflow
The Analysis App supports both mtDNA D‐Loop, custom regions, and whole genome
samples. See Analysis Methods, on page 28.
Nextera Adapter Trimming
• Nextera adapters are removed from the forward and reverse reads.
• Trimming is repeated until there are no more than 3 adapter bases on the end of the
read.
• Reads are discarded when they are excessively trimmed or have short amplicons.
Alignment
• BWA‐MEM performs the alignment.
• Circular alignment is handled across the origin by identifying the true start and end of
reads.
• Realigns regions with indels to improve alignment and shifts indels to a 3' alignment.
Account for Primers
• Manifest identifies the primers and amplicons.
• Removes primer contributions from the reads for accurate variant calling.
Variant Calling
• Identifies nucleotides that are greater than the interpretation threshold for the position.
• Generates percentage of reads for a nucleotide that exceeds the analysis threshold (at).
• Generates a percentage of reads for a nucleotide that exceeds the interpretation threshold
(it).
• Generates the interpretation threshold indicator for positions that have a nucleotide
between the analysis threshold and the interpretation threshold.
• Reports base call codes. See Base Call Codes, on page 31.
Filtering and Scoring
• Removes reads that do not meet the minimum quality score for a base call.
• Calculates a score from the minimum quality score for base call, alignment score, and
presence of low abundance reads at the position.
Output Files
• VCF files and BAM files.
• Use the mtDNA Variant Analyzer App to view the analysis results.
Log In to BaseSpace
1
Navigate to BaseSpace at basespace.illumina.com, enter your user name and password,
and click Login.
2
Click the Apps tab.
3
In Categories, click Targeted Sequencing, and then click mtDNA Variant Processor.
4
From the drop‐down list, select version 1.0.0, and then click Launch to open the app.
The mtDNA Variant Processor parameters window displays.
5
Set the parameters for analysis.
For additional training on BaseSpace, see the BaseSpace support page at
support.illumina.com/sequencing/sequencing_software/basespace/training.html
mtDNA Variant Processor BaseSpace App
Page 27
Set Analysis Parameters
1
In the App Session Name field, enter the analysis name.
By default, the analysis name includes the app name, followed by the date and time that
the analysis session starts.
2
From the Project field, select the project that stores the app results.
3
From the Samples field, browse to the sample you want to analyze, and select the
checkbox.
4
From the Minimum Base Call Quality Score for a Call (Q) field, enter the quality score.
The default value is 30. At each coordinate, reads at or above the Q value can contribute
to the variant call.
5
From the Analysis Threshold % field, enter the percentage.
The default value is 10. If a nucleotide call is at less than the value of the Analysis
Threshold (%), then the nucleotide call is not visible or reported as part of the call.
6
From the Interpretation Threshold % field, enter the percentage.
The default is 25. A nucleotide with a total number of reads greater than the
interpretation threshold percentage contributes to the call for the coordinate. When more
than one base exceeds this percentage for a coordinate, the app reports the base call code
for the bases. See Base Call Codes, on page 31.
7
From the Minimum Read Count field, enter the number of reads required at the
coordinate to produce a base call.
The default value 10.
8
From the Genome field, select the genome to be analyzed.
9
From the PCR Primer Description field, select from the following options:
• Whole Mito Genome Amplicon Manifest
• DLoop Amplicon Manifest
• Custom Manifest.
10 [Optional] If you select Custom Manifest, complete the following steps:
a
Enter the start coordinate (5', 0‐based) for the forward PCR primer.
b Enter the start coordinate (5', 0‐based) for the reverse PCR primer.
c
Enter the base length of the forward PCR primer.
d Enter the base length of the reverse PCR primer.
Click the plus (+) sign to add additional rows.
11 Click Continue.
The Analysis App begins analysis.
When analysis is complete, the app updates the status of the session and sends an email
to notify you.
mtDNA Variant Processor BaseSpace App
Page 28
Analysis Methods
The Analysis App uses the following methods to analyze the sequencing data. See Workflow
, on page 26.
• Illumina Secondary Analysis Software (Isas)
• BAM‐MEM 1
1
Li H. (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA‐MEM.
arXiv: 1303.3997v1[q‐bio.GN].
BWA‐MEM
BWA‐MEM is the most recent version of the Burrows‐Wheeler Alignment algorithm.
Optimized for longer read lengths of ≥ 70 bp, BWA‐MEM has a significant positive impact
on detection of variants, especially insertions and deletions.
Analysis Output
To view the results, click the Projects tab, then the project name, and then the analysis.
Figure 9
Analysis Output Navigation Bar
Use the left navigation bar to access the following analysis output:
• Analysis Info—Information about the analysis session, including log files.
• Inputs—Overview of input settings.
• Output Files—Output files for the samples.
Analysis Info
The Analysis Info page displays the analysis settings and execution details.
Row Heading
Definition
Name
Name of the analysis session.
Application
App that generated this analysis.
Date Started
Date and time the analysis session started.
Date Completed
Date and time the analysis session completed.
Duration
Duration of the analysis.
mtDNA Variant Processor BaseSpace App
Page 29
Row Heading
Definition
Session Type
Multi‐Node.
Status
Status of the analysis session. The status shows either Running or
Complete and the number of nodes used.
Log Files
File Name
Description
AmpliconCoverage_M#.tsv
Contains details about the resulting coverage per
amplicon per sample. M# represents the manifest
number.
AnalysisError.txt
Workflow standard error output (contains error
messages created when running the workflow).
AnalysisLog.txt
Workflow standard output (contains details about
workflow steps, command line calls with parameters,
timing, and progress).
MethylSeqRunStatistics.xml
Provides methylation‐related information.
SmallRNARunStatistics.xml
Provides methylation‐related information.
TargetedRNASeqRunStatistics.xml
Contains summary statistics specific to the run.
TST_15-A-Manifest.txt, TST_15-BManifest.txt
Manifest files used in the analysis.
Output Files
The Output Files page provides access to the output files for each sample analysis.
• BAM Files
• VCF Files
BAM File Format
A BAM file (*.bam) is the compressed binary version of a SAM file that is used to represent
aligned sequences up to 128 Mb. SAM and BAM formats are described in detail at
https://samtools.github.io/hts‐specs/SAMv1.pdf.
BAM files use the file naming format of SampleName_S#.bam, where # is the sample
number determined by the order that samples are listed for the run. In multi‐node mode, the
S# is set to S1, regardless the order of the sample.
BAM files contain a header section and an alignment section:
• Header—Contains information about the entire file, such as sample name, sample
length, and alignment method. Alignments in the alignments section are associated with
specific information in the header section.
mtDNA Variant Processor BaseSpace App
Page 30
• Alignments—Contains read name, read sequence, read quality, alignment information,
and custom tags. The read name includes the chromosome, start coordinate, alignment
quality, and the match descriptor string.
The alignments section includes the following information for each or read pair:
• RG: Read group, which indicates the number of reads for a specific sample.
• BC: Barcode tag, which indicates the demultiplexed sample ID associated with the read.
• SM: Single‐end alignment quality.
• AS: Paired‐end alignment quality.
• NM: Edit distance tag, which records the Levenshtein distance between the read and the
reference.
• XN: Amplicon name tag, which records the amplicon tile ID associated with the read.
BAM index files (*.bam.bai) provide an index of the corresponding BAM file.
VCF File Format
Variant Call Format (VCF) is a widely used file format developed by the genomics scientific
community that contains information about variants found at specific positions in a
reference genome.
Table 1
VCF Headings Table
Heading
Description
CHROM
The chromosome of the locus.
POS
The positional coordinate of the locus.
REF
The reference base for the POS and can only be A, C, T, G, or N.
ALT
The called base for the POS and can only be A, C, T ,G ,N, period (.), or
asterisk (*). The asterisk (*) is used when there is a deletion at the locus of an
upstream POS.
QUAL
The field is not used.
FILTER
PASS—For a called position
NoData—For a position without coverage.
INFO
The field is not used.
FORMAT
The field contains the key name for the information on each reported
attribute in the SAMPLE column. The keys are colon‐separated and
reference the #FORMAT descriptors in the header of the file.
SAMPLE
The field contains the values for each field from the FORMAT column.
mtDNA Variant Processor BaseSpace App
Page 31
Base Call Codes
The mtDNA Processor App supports variant calling for more than one nucleotide at a
position. When more than one nucleotide exceeds the interpretation threshold, the app
reports the IUPAC code as the consensus for the position.
Table 2
Variant Base Call Codes1 Table
Code
A
C
G
T
R
Y
S
W
K
M
B
D
H
V
N
‐
*
Base
Adenine
Cytosine
Guanine
Thymine
A or G
C or T
G or C
A or T
G or T
A or C
C or G or T
A or G or T
A or C or T
A or C or G
Any base
Deletion
No call
When data for the indel and a base call at a position exceed the interpretation threshold, the
app reports the ambiguous call code for an indel as the consensus for the position.
Table 3
1
Ambiguous Variant Base Call Codes Table
Code
a
c
g
g
r
y
s
w
k
m
b
d
h
v
n
Base
Adenine
Cytosine
Guanine
Thymine
a or g
c or t
g or c
a or t
g or t
a or c
c or g or t
a or g or t
a or c or t
a or c or g
Any base
Codes are from the International Union of Pure and Applied Chemistry (IUPAC).
mtDNA Variant Processor BaseSpace App
Page 32
For example, Figure 10 shows the presence of an ambiguous deletion at position 501.
Because there are an equal number of reads that support a deletion call and a reference call
of G, the app reports an ambiguous call of g as the consensus.
Figure 10
Example of Ambiguous Variant Base Calls
mtDNA Variant Analyzer BaseSpace App
Page 33
mtDNA Variant Analyzer BaseSpace App
Using the analysis results from the mtDNA Variant Processor App, the BaseSpace® mtDNA
Variant Analyzer App produces the analysis for mitochondrial DNA (mtDNA) sequencing
data. The app can analyze any part of the full circular genome, without any origin dead
zone, using quality and coverage thresholds customized by the user. The app also generates
analysis reports in Excel file format.
See the mtDNA Variant Analyzer App Guide (document # 1000000007934) for complete
information on the application.
Compatible Data
The Variant Call Format (VCF) output files from the mtDNA Variant Processor App are
compatible with the Analysis App.
Reference Genomes
The genome for alignment is the Revised Cambridge Reference Sequence (rCRS), a mtDNA
genome used by the Forensic Genomics community.
Log In to BaseSpace
1
Navigate to BaseSpace at basespace.illumina.com, enter your user name and password,
and click Login.
2
Click the Apps tab.
3
In Categories, click Targeted Sequencing, and then click mtDNA Variant Analyzer.
4
From the drop‐down list, select version 1.0.0, and then click Launch to open the app.
The permission request dialog box appears.
5
From the dialog box, click Accept.
By default, this dialog box appears the first time you launch the app. After you click
Accept, the BaseSpace Projects page appears.
For additional training on BaseSpace, see the BaseSpace support page at
support.illumina.com/sequencing/sequencing_software/basespace/training.html
BaseSpace Projects Page Functions
You can perform the following functions while in the BaseSpace Projects page:
• To search for a project, enter the project name in the search project name field.
• To search for samples, enter a sample name in the search sample name field.
• To remove a sample, click the selected sample again or click
• To select all samples, click the Select All Visible link.
• To remove all samples, click Clear all.
.
mtDNA Variant Analyzer BaseSpace App
Page 34
Select Samples From a Project or Multiple Projects
1
In the BaseSpace Projects page, select the project you want to analyze.
2
Select the samples you want to analyze.
The app supports a maximum of 96 samples. The right‐side pane shows the selected
samples for analysis.
3
[Optional] To add samples from a different project, click BaseSpace Projects to return to
the BaseSpace projects page.
Repeat step 1 and 2.
4
When you have completed selecting the samples, click Submit.
By default, the first time you select a sample, a dialog box requesting for access to
samples in the project appears.
5
From the dialog box, click Accept
The app reloads the selected samples. Any unsaved changes to samples are not loaded
to the analysis. When analysis is complete, the app shows the sample analysis in the
Reference Genome Compare View, mtDNA Navigator, and Coverage Plot.
Analysis Output
The mtDNA Variant Analyzer App uses VCF and BAM output files from the mtDNA
Variant Processor App to produce the following analysis output:
• Reference Genome Compare View
• Sample Compare View
• mtDNA Navigator
• Coverage Plot
• Aggregate Analysis Report
• Sample Analysis Reports
Status Icons
The Analysis App provides the following status icons.
Table 4
Icon
Status Icon Table
Description
In the BaseSpace Projects page, this icon at the top‐right
of the sample indicates that the sample is modified.
In the Reference Genome Compare View, this blue icon
next to the sample indicates that the sample is modified.
In the Sample Compare View, the icon color is orange.
In the Reference Genome Compare View, this blue icon
next to the sample name indicates that the sample
changes are unsaved.
In the Sample Compare View, the icon color is orange.
mtDNA Variant Analyzer BaseSpace App
Page 35
Reference Genome Compare View
The Reference Genome Compare View has a blue background and lets you compare the
reference genome (rCRS) with the selected samples. You can view the following information
in the view pane:
• Reference genome
• Selected samples for comparison
• Call of a position for each sample
• Variant indicator (blue line under the call)
• Interpretation threshold indicator (purple line under the call)
• User modified call indicator (asterisk sign next to the call)
• Total number of samples
Any changes you make are immediately updated to the following areas:
• Reference Genome Compare View
• mtDNA Navigator
• Coverage Plot
When you select a sample (it is highlighted), the mtDNA Navigator and Coverage Plot
immediately update the position and coverage for that sample.
Use these tools to refine your analysis. When you make changes to the Reference Genome
Compare View, the changes are also reflected in the mtDNA Navigator and Coverage Plot.
Table 5
Tools
Reference Genome Compare View Tools Table
Description
Sample Compare—To make a selected sample a
reference and compare it to the rest of the samples,
highlight the sample by selecting it, and then drag the
blue dot to the other side until the background is orange.
Use this tool to find differences between samples or to
find identical samples.
Sample Selection—To add more samples from the same
project or a different project, use this tool to return to the
BaseSpace projects page.
Save—When you modify a base call, use this tool to save
changes to BaseSpace. The icon is activated when you
make a change. Click Sample Selection to view samples
that are modified.
Filter—Use this tool to filter samples for D‐Loop,
variants, interpretation threshold, and user modified. See
Filter Options, on page 36.
Call for a Position—Use this tool to view call details. See
View Call Details, on page 37.
Generate Report—Use this tool to export sample analysis
data in Excel file format.
Next Hotspot—A hotspot is any variant, interpretation
threshold indicator, or user modified position for the
selected sample. Use the large arrow to get to the next
hotspot.
Next Page—Use the small arrow to get to the next 15
positions.
mtDNA Variant Analyzer BaseSpace App
Page 36
Filter Options
You can select one or more filters per analysis.
Table 6 Reference Genome Compare Filter Options Table
Filter
Description
D‐Loop
This option constrains the view range positions from
16024 to 576.
The constraint affects the Reference Genome Compare
View, mtDNA Navigator, and Coverage Plot.
Variants
This option filters the positions with variants for each
sample.
Interpretation Threshold
This option filters the positions with interpretation
threshold indicators for each sample.
User Modified
This option filters the positions that are modified.
Sample Compare View
The Sample Compare View has an orange background and lets you compare a specific
sample with the selected samples. The sample located at the top of the list is the reference
sample. You can view the following information in the view pane:
• Reference sample
• Selected samples for comparison
• Call of a position for each sample
• Differences indicator (orange line under the call)
• Interpretation threshold indicator (purple line under the call)
• User modified call indicator (asterisk sign next to the call)
• Total number of samples
Any changes you make are immediately updated to the following areas:
• Sample Compare View
• mtDNA Navigator
• Coverage Plot
When you select a sample (it is highlighted), the mtDNA Navigator and Coverage Plot
immediately update the position and coverage for that sample and the reference sample.
The tools for tools for the Reference Genome Compare View and Sample Compare View are
the same, with the exception of the following differences. See Reference Genome Compare
View, on page 35.
Table 7
Tools
Sample Compare View Tools Table
Description
Sample Compare—To return to the Reference Genome
Compare View, drag the orange dot to the other side
until the background is blue.
Filter—Use this tool to filter samples for D‐Loop,
differences and zero differences, interpretation threshold,
and user modified. See Filter Options, on page 37.
Sample Navigation—Use this tool change the sample
you want to compare. Click the arrow to move the
selected sample to the top of the list.
mtDNA Variant Analyzer BaseSpace App
Page 37
Filter Options
You can select one or more filters per analysis.
Table 8 Sample Compare Filter Options Table
Filter
Description
D‐Loop
This option constrains the view range positions from
16024 to 576.
The constraint affects the Sample Compare View,
mtDNA Navigator, and Coverage Plot.
Differences
This option filters the positions that are different from the
reference sample.
Interpretation Threshold
This option filters the positions with interpretation
threshold indicators for each sample.
User Modified
This option filters the positions that are modified.
Zero Differences
This option filters samples that are not different from the
reference sample.
View Call Details
to view the Detailed Call View.
1
Click a specific call position
2
[Optional] From the Original Call field, expand the drop‐down arrow to select a different
call.
Detailed Call View
Table 9
Detailed Call View Table
Call Detail
Description
Reference Sample Name
The reference sample name.
Sample Name
The sample name for the result.
Call
The call for the position.
Paired-End Depth
The maximum depth of either the forward or reverse reads.
Score
Score for the call.
Percentages
The percentages are stacked and shown in relation to the
thresholds.
it
The location where the interpretation threshold resides for the
sample.
at
The location where the analytical threshold resides for the sample.
Original Call
The original call. To change the call, expand the drop‐down arrow
and select a call from the list.
mtDNA Variant Analyzer BaseSpace App
Page 38
mtDNA Navigator
The following are the mtDNA Navigator features:
• The pointer indicates the position in the Reference Genome Compare View or Sample
Compare View.
• To view genomic variants, interpretation threshold, D‐Loop positions, and selected
coverage, move the pointer around the circle plot.
• The mtDNA Navigator zooms in to show more details of the D‐Loop area when you
select the D‐Loop filter option in the Reference Genome Compare View or Sample
Compare View.
• The mtDNA Navigator shows coverage for the selected sample in relation to the visual
representation of the genome. In Sample Compare View, the app shows the overlapped
coverage of the reference sample and selected sample.
Any changes you make are immediately updated in the following areas:
• mtDNA Navigator
• Reference Genome Compare View or Sample Compare View
• Coverage Plot
Coverage Plot
The following are the Coverage Plot features:
• The pointer indicates the position in the Reference Genome Compare View or Sample
Compare View.
• To zoom in and out, enter a value in the Coverage field.
• When you are in Reference Genome Compare View, the blue line in the coverage plot
represents the coverage for the selected sample.
• When you are in Sample Compare View, the gray line in the coverage plot represents the
coverage for the reference sample and the orange line represents the coverage for the
selected sample.
Any changes you make are immediately updated in the following areas:
• Coverage Plot
• Reference Genome Compare View or Sample Compare View
• mtDNA Navigator
Aggregate Analysis Report
The Analysis App generates an aggregated summary of all the analyzed samples in an Excel
file.
Table 10
Summary Table
Statistic
Definition
Sample
The sample name.
Single Nucleotide
Variants (SNVs)
The number of positions where the call for the sample differs from
the reference genome (rCRS).
mtDNA Variant Analyzer BaseSpace App
Page 39
Statistic
Definition
Insertions
The number of one or more nucleotides inserted in the sample
genome.
Deletions
The number of one or more nucleotides deleted from the sample
genome.
Manually Changed Calls
The changed call when it is modified, which is presented as
[position] [original call] ‐> [new call].
Variants
The position and call for calls that differ from the reference
genome.
Sample Analysis Reports
In addition to the aggregated summary of all the analyzed samples, the Analysis App
generates a summary of read count and variant statistics for each sample in the same Excel
file.
Table 11
Sample Analysis Table
Statistic
Definition
Sample
The sample name.
Single Nucleotide
Variants
The number of positions where the call for the sample differs from
the reference genome (rCRS).
Insertions
The number of one or more nucleotides inserted in the sample
genome.
Deletions
The number of one or more nucleotides deleted from the sample
genome.
Manually Changed Calls
The changed call when it is modified, which is presented as
[position] [original call] ‐> [new call].
Variant
The position and call for calls that differ from the reference
genome.
Reference
The base position in the reference genome (rCRS).
When there is an insertion, the reference base reports a plus (+)
sign.
Score
The score for the call.
Paired-End Depth
The maximum depth of either the forward or reverse reads.
Reads A, C, G, T
The number of forward and reverse reads for the A, C, G, or T
bases at the call position.
Percent A, C, G, T
The percentage of forward and reverse reads out of the total reads
for the A, C, G, or T bases at the call position.
mtDNA Variant Analyzer BaseSpace App
Page 40
Statistic
Definition
Reads Del
The number of forward and reverse reads for a deletion at the call
position.
Percent Del
The percentage of forward and reverse reads out of the total reads
for a deletion at the call position.
MiSeq Reporter
Page 41
MiSeq Reporter
The mtDNA MiSeq Reporter Plug‐In and the mtDNA Variant Analyzer are exclusively
designed for mtDNA samples run on a MiSeq or MiSeq FGx instrument. This section
describes the installation and output of these mtDNA analysis tools.
These analysis tools are only compatible with MCS v2.3 used with MiSeq Reporter v2.3, or
FGx Control Software v1.1 used with MiSeq Reporter 2.5.1.3. Other software configurations
are not guaranteed to be compatible and are used at risk. Contact your Illumina
representative for more information.
NOTE
The following procedures assume that MiSeq Reporter is installed on your instrument control
computer, you are familiar with how to run MiSeq Reporter, and have read the MiSeq Reporter
Software Guide (document # 15042295).
If using MiSeq Reporter for analysis, a sample sheet is required before starting the run. See
Create a Sample Sheet, on page 42. Make sure that a mtDNA Genome manifest provided by
Illumina is present in the MiSeq run directory before the initiation of MiSeq Reporter
analysis. Edit the manifest if the primers for your experiment differ from the primers
recommended in this protocol. See Edit Manifest, on page 43
Install mtDNA MiSeq Reporter Plug‐In
The mtDNA MiSeq Reporter Plug‐In is provided as a software plug‐in for MiSeq Reporter. It
is designed for processing human mtDNA samples for use with the mtDNA Variant
Analyzer. Install the plug‐in as follows:
1
Download the MiSeq Reporter plug‐in installer and copy the installer file to a location
accessible from the MiSeq instrument computer.
NOTE
• If you are using MiSeq Reporter off‐instrument, the mtDNA MiSeq Reporter Plug‐In
must be installed and run on a computer that has MiSeq Reporter installed.
• If another version of the mtDNA MiSeq Reporter Plug‐In is installed, uninstall it using
the Windows Control Panel before installing the current version.
2
Double‐click the MiSeq Reporter plug‐in installer (mtDNA Miseq Reporter Plug‐In.msi)
and follow the software prompts to complete installation.
3
Copy the rCRS1 folder in the installation package to the genomes folder accessed by
MiSeq reporter (C:\illumina\MiSeq Reporter\Genomes).
1
Andrews R, Kubacka I, Chinnery P, Lightowlers R, Turnbull D, and Howell N (1999) Reanalysis
and revision of the Cambridge reference sequence for human mitochondrial DNA. Nature
Genetics 23: 147.
Install mtDNA Variant Analyzer
The mtDNA Variant Analyzer opens the data in the viewer files (.viewer) produced by the
mtDNA MiSeq Reporter Plug‐In. Install the analyzer as follows:
MiSeq Reporter
Page 42
NOTE
• The mtDNA Variant Analyzer requires Microsoft Office 2010 and Microsoft.NET
framework version 4.0.
• Install the mtDNA Variant Analyzer on a computer with access to the output from the
mtDNA MiSeq Reporter Plug‐In.
• The *.viewer files are the only files required by the mtDNA Variant Analyzer. The files for
the samples of interest can be copied to any network location or locally for portability.
1
Download the mtDNA Variant Analyzer installation package and copy the installer file
to a computer.
NOTE
If another version of the mtDNA Variant Analyzer is installed, uninstall it using the
Windows Control Panel before installing the current version.
2
Double‐click the mtDNA Variant Analyzer installer (mtDNA Setup.msi) and follow the
software prompts to complete installation.
Create a Sample Sheet
1
Create a sample sheet using the Illumina Experiment Manager (IEM) software.
2
After creating the sample sheet, open it in Microsoft Excel and modify the following
fields for mtDNA analysis:
• Header—Enter “mtDNA” in the Workflow row.
• Settings—Enter the Nextera XT adapter sequence, used in the assay, in the Adapter row.
• Manifests—Identify the PCR Amplicon manifest used for the assay. Each manifest is an
independent entry in this section and referenced by a letter.
NOTE
If the primers for your experiment differ from those recommended in this protocol, edit the
manifest. See Edit Manifest, on page 43. Make sure that the sample sheet correctly references
the manifest for the samples amplified with the custom primers.
Make sure that the sample sheet correctly references the manifest for the samples amplified
with the custom primers. See
• Data—Each sample must refer to the appropriate manifest defined in the Manifests
section. Each sample must reference the rCRS GenomeFolder.
3
[Optional] Define additional settings that alter the behavior of variant calling in the
Settings section of the sample sheet:
NOTE
If the following settings are modified, requeue the run for analysis in MiSeq Reporter. For
more information, see the MiSeq Reporter User Guide (part # 15042295).
• MinimumBasecallQualityForVariantCall—At each coordinate, the reads contributing to
the variant call must be at or above this Q value to contribute to the call. If this value is
not specified, the default is 30 (Q30).
• DetectionThreshold—Variants less than this value will not be reported. The value can
be specified. If the value is not specified, the default is 0.10 (10%).
• AnalysisThreshold— A called base with a frequency greater than this value is reported
as the code between the base and the reference. If this value is not specified, the default
is 0.25 (25%, variant frequencies between 25% – 75% will be considered ambiguous). For
more information, see Ambiguous Variant Base Calls, on page 50.
MiSeq Reporter
Page 43
4
Save the sample sheet with the name SampleSheet.csv and place it in the MiSeq run
directory (D:\Illumina\MiSeq FGx Control Software\SampleSheets).
Figure 11
mtDNA Genome Sample Sheet Example
Edit Manifest
The MiSeq Reporter Plug‐In requires a mtDNA Genome manifest that defines the location of
primers.
If the primers for your experiment differ from the primers recommended in this protocol, edit
the manifest to reflect the regions amplified as follows:
1
Open the mtDNA Genome manifest provided by Illumina using Microsoft Excel.
2
Edit the Name, Amplicon Start, Amplicon End, Upstream Probe Length, and
Downstream Probe Length for a contiguous amplified region.
3
For amplicons that span the origin, enter an amplicon that ends at position 16569 with a
length of 1, and another amplicon that begins at position 1 with a length of 1.
4
Save the manifest in the manifest folder in the directory (D:\Illumina\MiSeq FGx
Control Software\Manifests).
NOTE
• If you change the file name of the manifest, make sure that your sample sheet
references the new name for each sample.
• After sequencing begins, the manifest is copied to the run directory of the run. If
reprocessing a run with a different manifest, revise or replace the manifest that is
within the run directory.
MiSeq Reporter Analysis Output
After the mtDNA MiSeq Reporter Plug‐In is installed, perform mtDNA secondary analysis
by following MiSeq Reporter standard operations. For more information, see the MiSeq
Reporter Software Guide (part # 15042295).
MiSeq Reporter
Page 44
The mtDNA MiSeq Reporter Plug‐In creates an output file (.viewer) for the mtDNA Variant
Analyzer. Each coordinate position that had supporting data for the sample is described
with:
•
•
•
•
•
•
The reference base at the position
The call at the position. When the call is identical to the reference, a “.” is presented.
The mean base quality score at the position
The variant score at the position
The number of reads contributing to each base call
Any flags highlighting issues with the position. The list of possible choices are as
follows:
Table 12 Potential Position Flags for Secondary Analysis Output
Issue
Definition
LowVariantFreq Variant frequency is lower than the cut‐off (default 0.10)
LowGQ
Variant quality score was below the cut‐off (30.00)
IndelRepeatLength Insertion or Deletion length is greater than the cut‐off (8)
LowDP
Coverage depth lower than cut‐off (10)
SB
Variant strand bias too high or coverage on only 1 strand
DEL
Filter is related to a deletion or flanking deletion
ChkTSNP
Check T SNP call in C‐stretch (position 310)
ChkMultiCall
Check multiple calls at single position
mtDNA Variant Analyzer
The mtDNA Variant Analyzer compares a Question sample to a Known sample or the rCRS
reference using the results from MiSeq Reporter running with the mtDNA MiSeq Reporter
Plug‐In. The software workflow has 3 consecutive processes:
• Identify Samples
• Interrogate Data
• Report
Start Variant Analyzer
After completing the installation, start the mtDNA Variant Analyzer.
1
From the Windows Start menu, click All Programs | Illumina | mtDNA Variant
Analyzer or double‐click the mtDNA Variant Analyzer
The mtDNA Variant Analyzer screen appears.
icon on your desktop.
MiSeq Reporter
Page 45
Figure 12
mtDNA Variant Analyzer Screen | Identify Samples Tab
Identify Samples
Identify a known and question sample, for comparative analysis, on the Identify Samples tab
of the mtDNA Variant Analyzer screen:
1
Do 1 of the following to select the Known sample for comparison to the Question
sample:
• Click Browse to specify a sample (.viewer file) from the Alignment folder in the
MiSeqAnalysis folder (Data\Intensities\BaseCalls\Alignment).
• Select the Compare to rCRS checkbox for rCRS comparison.
2
Click Browse to select the Question sample (.viewer file) from the Alignment folder in
the MiSeqAnalysis folder (Data\Intensities\BaseCalls\Alignment).
3
Click Next.
Interrogate Data
Data from the selected samples are displayed on the Interrogate tab of the mtDNA Variant
Analyzer screen.
MiSeq Reporter
Page 46
Figure 13
Example: Interrogate Tab
Coverage Plot
A coverage plot is displayed at the top of the screen.
• When 2 samples are selected for comparison, both are represented in the plot.
• The Known sample is represented in orange.
• The Question sample is represented in gray.
• If the rCRS is selected for comparison to the Question sample, only the Question plot
is represented in gray.
• Select the Only show D-Loop checkbox to resize the base coordinate axis to display only
the D‐loop.
The range of coordinates that are visible in the sequence display is highlighted in green
on the coverage plot.
• Drag the green highlighted area to adjust the coordinates that are visible in the sequence
section.
Sequence Data
The sequence data are displayed in the center of the screen.
• The Known or rCRS and Question coordinate and base calls are displayed.
• Drag the slide bar to traverse the coordinates.
The green highlighted area in the coverage plot is simultaneously adjusted to cover the
coordinates that are visible in the sequence section.
• Click a sequence coordinate to view additional information about that coordinate in the
Detail View at the bottom of the screen. The selected sequence coordinate is outlined in
yellow.
MiSeq Reporter
Page 47
Figure 14
Example: Sequence Detail View
• A base is shaded gray when there is more than 1 nucleotide with supporting reads.
• Each base is color coded according to the Mean Basecall Quality Score of the reads
supporting the call.
• Click Settings to edit the base color thresholds.
Figure 15
Settings
• To edit a call manually, double‐click the base and enter a new value. This change is
tracked and reported during the reporting phase. See Create a Sample Sheet, on page 42
for information on acceptable input when changing a sample.
• Select the Only show differences checkbox to resize the base coordinate axis to display
only the differences between the samples.
• When comparing a Known sample and a Question sample, only the differences between
the samples are displayed, regardless of differences from the rCRS.
• When comparing a Question sample to the rCRS, only the Question sample’s variants
are displayed.
MiSeq Reporter
Page 48
Do 1 of the following:
• Click Next to proceed to the Report tab.
• Click Back to return to the Identify Samples tab.
Report
A summary of the results of the interrogated samples is displayed on the Report tab of the
mtDNA Variant Analyzer screen.
Figure 16
Example: Report Tab
The following parameters are provided for each sample:
• Number of single nucleotide variants
• Number of insertions identified
• Number of deletions identified
• A summary of calls changed manually in the Interrogate tab
• A summary of the variants
The interrogation report can be saved.
1
Do 1 of the following:
• Select Export Excel to save a summary of the variant information in an Excel (*.xlsx)
format.
• Select Export HSD to save a summary of the variant information in a *.txt format for
import into applications, such as HaploGrep1.
1
Kloss‐Brandstaetter A., Pacher D., Schoenherr S., Weissensteiner H., Binna R., Specht G.,
Kronenberg F. 2010 HaploGrep: a fast and reliable algorithm for automatic classification of
mitochondrial DNA haplogroups www.haplogrep.uibk.ac.at doi: 10.1002/humu.21382 van
Oven M, Kayser M. 2009. Updated comprehensive phylogenetic tree of global human
mitochondrial DNA variation. Hum Mutat 30(2):E386–E394. www.phylotree.org .
doi:10.1002/humu.20921.
MiSeq Reporter
Page 49
2
•
•
•
•
•
When prompted, name the file and browse to a location to save the file.
A saved report contains the information on the display and the following additional
information for each variant:
Mean basecall quality score
Variant score
Paired‐end depth
Total number of reads supporting each base contributing to the call
Percentage contribution for each base contributing to the call
NOTE
In a saved report, the total number of Reads A, Reads C, Reads G, and Reads T does not
equal the Paired‐End Depth. The Paired‐End Depth is the maximum of either the forward
or reverse reads.
3
Do 1 of the following:
• Select the Windows close
to exit mtDNA Variant Analyzer.
• Select Back to return to the Interrogate tab.
Supporting Information
Page 50
Supporting Information
The protocols described in this guide assume that you have reviewed the contents of this
section, confirmed your kit contents, and obtained all the required consumables and
equipment.
Ambiguous Variant Base Calls
The mtDNA MiSeq Reporter Plug‐In supports nucleotide analysis and variant calling for up
to two bases at any given position. When a base is called with a frequency greater than the
interpretation threshold, the base call is reported as a code between the base and the
reference (Table 13).
Table 13
Variant Base Call Codes1
Code
A
C
G
T
R
Y
S
W
K
M
‐
Base
Adenine
Cytosine
Guanine
Thymine
A or G
C or T
G or C
A or T
G or T
A or C
Deletion
When a base call contains reads that pass the interpretation threshold, and there are
sufficient number of reads passing the interpretation threshold both with and without an
indel, the call is indicated in the analysis data as an ambiguous indel code.
Table 14 Ambiguous Variant Base Call Codes
Code
Base
c
Ambiguous Insertion
t
Ambiguous Insertion
a
Ambiguous Insertion
g
Ambiguous Insertion
.
Ambiguous Deletion
1
Codes are from the International Union of Pure and Applied Chemistry (IUPAC).
Supporting Information
Page 51
Acronyms
Table 15
Acronyms
Acronym
Definition
ATM
Amplicon Tagment Mix
DAL
Diluted Amplicon Library
IEM
Illumina Experiment Manager
LNA1
Library Normalization Additives 1
LNB1
Library Normalization Beads 1
LNP
Library Normalization Plate
LNS1
Library Normalization Storage Buffer 1
LNW1
Library Normalization Wash 1
MgCl2
Magnesium Chloride
mtDNA
mitochondrial DNA
N7XX
Adapter Index
NPM
Nextera PCR Master Mix
NT
Neutralize Tagment Buffer
NTA
Nextera XT Tagment Amplicon Plate
PAL
Pooled Amplicon Library
rCRS
Cambridge Reference Sequence
RSB
Resuspension Buffer
S5XX
Adapter Index
SGP
Storage Plate
TD
Tagment DNA Buffer
Consumables and Equipment
Make sure that you have the required user‐supplied consumables and equipment before
starting the protocol.
The protocol has been optimized and validated using the items listed. Comparable
performance is not guaranteed when using alternate consumables and equipment.
Supporting Information
Page 52
Consumables
Consumable
Supplier
10 µl barrier pipette tips
General lab supplier
10 µl multichannel pipettes
General lab supplier
10 µl single channel pipettes
General lab supplier
15 ml conical tubes
General lab supplier
20 µl barrier pipette tips
General lab supplier
20 µl multichannel pipettes
General lab supplier
20 µl single channel pipettes
General lab supplier
2.5 L Ice bucket
General lab supplier
96‐well PCR plates
General lab supplier
96‐well storage plates, round well, 0.8 ml
("MIDI" plate)
Fisher Scientific, catalog # AB‐0859
200 µl barrier pipette tips
General lab supplier
200 µl multichannel pipettes
General lab supplier
200 µl single channel pipettes
General lab supplier
1000 µl barrier pipette tips
General lab supplier
1000 µl multichannel pipettes
General lab supplier
1000 µl single channel pipettes
General lab supplier
Agencourt AMPure XP 5 ml kit
Beckman Coulter Genomics, item # A63880
Eppendorf tubes (screw cap recommended)
General lab supplier
Ethanol 200 proof (absolute)
for molecular biology (500 ml)
Sigma‐Aldrich, product # E7023
Microseal 96‐well PCR plates (“TCY” plate)
Bio‐Rad, catalog # HSP‐9601
Microseal ‘A’ film
Bio‐Rad, catalog # MSA‐5001
Microseal ‘B’ adhesive seals
Bio‐Rad, catalog # MSB‐1001
One of the following kits:
• MiSeq Reagent Kit v2 (300 cycle)
• MiSeq Reagent Kit v3 (600 cycle)
• MiSeq FGx Reagent Kit v3 (600 cycle)
Illumina, catalog #:
• MS‐102‐2002
• MS‐102‐3001
• TG‐143‐1001
Supporting Information
Page 53
Consumable
Supplier
One of the following kit pairs:
• Nextera XT DNA Library Prep Kit (24
samples) and
• Nextera XT DNA Library Prep Index Kit (24
Indices, 96 Samples)
or
• Nextera XT DNA Library Prep Kit (96
samples) with one of the following:
• Nextera XT DNA Library Prep Index
Kit v2 Set A (96 Indices, 384 Samples)
• Nextera XT DNA Library Prep Index
Kit v2 Set B (96 Indices, 384 Samples)
• Nextera XT DNA Library Prep Index
Kit v2 Set C (96 Indices, 384 Samples)
• Nextera XT DNA Library Prep Index
Kit v2 Set D (96 Indices, 384 Samples)
Illumina, catalog #:
• FC‐131‐1024 and
Nuclease‐free water
General lab supplier
PCR 8‐tube strips with caps
General lab supplier
PhiX Control v3
Illumina, catalog # FC‐110‐3001
Purified Human mtDNA (100 pg/µl)
User experimental samples
TaKaRa LA Taq DNA Polymerase,
10X LA PCR Buffer II, and
2.5 mM dNTP Mix
TaKaRa, catalog # RR002M
TruSeq Index Plate Fixture Kit
Illumina, catalog # FC‐130‐1005
Tris‐HCl 10 mM, pH8.5
(For Bioanalyzer‐based normalization only)
General lab supplier
Tween 20
(For Bioanalyzer‐based normalization only)
Sigma, product # P7949
• FC‐131‐1001
• FC‐131‐1096 with one of following:
• FC‐131‐2001
• FC‐131‐2002
• FC‐131‐2003
• FC‐131‐2004
PCR Primers
Obtain the following PCR Primers1 from a general oligo supplier.
Supporting Information
Page 54
Primer
Sequence
MTL‐F1
5’‐ AAA GCA CAT ACC AAG GCC AC ‐3’
MTL‐F2
5’‐ TAT CCG CCA TCC CAT ACA TT ‐3’
MTL‐R1
5’‐ TTG GCT CTC CTT GCA AAG TT ‐3’
MTL‐R2
5’‐ AAT GTT GAG CCG TAG ATG CC ‐3’
1
Stawski, H., B. J. Bintz, E. S. Burnside, and M. Wilson. 2013. Preparing Whole Genome Human
Mitochondrial DNA Libraries for Next Generation Sequencing (NGS) Using Illumina Nextera XT.
Poster presentation at the 65th Annual American Academy of Forensic Sciences Conference. In:
Proceedings of the American Academy of Forensic Sciences. Washington, D.C.
www.aafs.org/sites/default/files/pdf/ProceedingsWashingtonDC2013.pdf
Equipment
Equipment
Supplier
96‐well thermal cycler with heated lid
General lab supplier
2100 Bioanalyzer Desktop System
Agilent, catalog # G2940CA
Agilent DNA 12000 Kit
Agilent, catalog # 5067‐1508
[Optional] Agilent High Sensitivity DNA Kit
Agilent, catalog # 5067‐4626
[Optional] One of the following microplate shakers for bead‐
based normalization:
• BioShake XP High‐Speed Mixer
• VWR Advanced High‐Speed Microplate Shaker
• VWR Signature High‐Speed Microplate Shaker
• Q Instruments, model # 1808‐
0505
• VWR, catalog # 14216‐214
(230 V)
• VWR, catalog # 13500‐890
(110 V/120 V)
Magnetic stand‐96
Thermo Fisher Scientific,
catalog # AM10027
One of the following Illumina sequencing instruments:
• MiSeq
• MiSeq FGx
Illumina, catalog #: • SY‐410‐1003
• SY‐411‐1001
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement