PreHandbook Template Word 2010

PreHandbook Template Word 2010
June 2015
GeneRead™ DNAseq Targeted Panels
V2 Handbook
For targeted enrichment prior to nextgeneration sequencing
(96-well plate compatible, all-bead purification)
Sample & Assay Technologies
QIAGEN Sample and Assay Technologies
QIAGEN is the leading provider of innovative sample and assay technologies,
enabling the isolation and detection of contents of any biological sample. Our
advanced, high-quality products and services ensure success from sample to
result.
QIAGEN sets standards in:

Purification of DNA, RNA and proteins

Nucleic acid and protein assays

microRNA research and RNAi

Automation of sample and assay technologies
Our mission is to enable you to achieve outstanding success and breakthroughs.
For more information, visit www.qiagen.com.
Contents
Kit Contents
5
Storage
7
Intended Use
8
Safety Information
8
Quality Control
8
Introduction
9
Principle and procedure
9
Equipment and Reagents to be Supplied by User
12
Important Notes
14
DNA preparation and quality control
14
DNA quantification and quality control
14
Protocol: PCR Setup
16
Protocol: Sample Pooling and Purification
22
Troubleshooting Guide
24
References
24
Appendix A: Library Construction Using GeneRead Library Prep Kits for
Illumina
25
End repair of DNA
25
A-addition
26
Adapter ligation
26
Cleanup of adapter-ligated DNA with AMPure XP beads
27
PCR amplification of purified library
28
Cleanup of amplified library with AMPure XP beads
29
Appendix B: Library Construction Using GeneRead Library Prep Kits for Ion
PGM Sequencer/Proton
31
End repair of DNA
31
Adapter ligation
32
Cleanup of adapter-ligated DNA with AMPure XP beads
33
PCR amplification of the purified library
34
Cleanup of amplified library with AMPure XP beads
35
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
3
Appendix C: FFPE DNA Quality and Quantity
37
Appendix D: Library Quantification and Quality Control
38
Appendix E: Data Analysis using QIAGEN’s GeneRead DNAseq Sequence
Variant Analysis Software
39
Appendix F: Combine Libraries for Multiplex Sequencing
40
Appendix G: Analyze the PCR amplicons and Library Using the QIAxcel
Advanced with a QIAxcel DNA High Resolution Kit or Agilent 2100
Bioanalyzer
41
Appendix H: Downloading Individual Unaligned .BAM File with a Multiplex
Sample on Ion PGM Sequencer
43
Ordering Information
4
45
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Kit Contents
GeneRead DNAseq Targeted Panel V2
Catalog no.
Pools with enough primers for 12 or 96 samples,
depending on pack size
Handbook
181900*
1†/4
1
* Gene panel pools are labeled A1, A2, A3 and A4.
†
E.g., the Tumor Actionable Mutations GeneRead DNAseq Targeted Panel V2.
GeneRead DNAseq Targeted HC Panel V2
‡
§
Pools with enough primers for 12 or 96 samples,
depending on pack size
4
Handbook
1
Gene panel pools are labeled A1, A2, A3 and A4.
E.g., the Human Comprehensive Cancer, Carrier Testing and Cancer Predisposition
GeneRead DNAseq Targeted HC Panel V2.
GeneRead DNAseq Custom Panel V2
Pools with primers for any gene or region in the human
genome, up to 9600 primer pairs, for 480 samples
Handbook
¶
181901§‡
181902
1/2/3/4¶
1
Number of pools determined by covered region.
GeneRead DNAseq Mix-n-Match Panel V2
181905
Pools with bench-tested primers for a mix of any gene
in catalog panels, up to 9600 primer pairs, for 96
samples
4
Handbook
1
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
5
Catalog no.
Product name
No. of total
primer pairs
No. of pools
NGHS-001X
Human Breast Cancer Panel
2915
4
NGHS-002X
Human Colorectal Cancer Panel
1954
4
NGHS-003X
2536
4
NGHS-004X
Human Myeloid Neoplasms
Panel
Human Liver Cancer Panel
2052
4
NGHS-005X
Human Lung Cancer Panel
3586
4
NGHS-006X
Human Ovarian Cancer Panel
2021
4
NGHS-007X
Human Prostate Cancer Panel
1837
4
NGHS-008X
Human Gastric Cancer Panel
2377
4
NGHS-009X
Human Cardiomyopathy Panel
2657
4
NGHS-011X
Human Carrier Testing Panel
6943
4
NGHS-013X
Human Cancer Predisposition
Panel
Human Comprehensive Cancer
Panel
Clinically Relevant Tumor Panel
6582
4
7951
4
602
4
Human BRCA1 and BRCA2
Panel
Tumor Actionable Mutations
Panel
250
4
118
1
NGHS-501X
NGHS-101X
NGHS-102X
NGHS-201X
6
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
GeneRead DNAseq Panel PCR
Kit V2
181940
181942
(12)
(96)
1
(24/24/16/12*
samples)
(192/192/128/96*
samples)
GeneRead DNAseq Panel 5x
PCR Buffer
230 µl
1800 µl
GeneRead HotStarTaq® DNA
Polymerase 6 U/µl
80 µl
600 µl
1000 µl
1000 µl
DNase-free water
* Number of samples that can be processed for 1, 2, 3 or 4 pool panels, respectively.
Required mastermix quantities depend on the number of PCR pools and number
of samples.
# PCR pools
1
2
3
4
# samples per
panel
12
96
480
480
480
12
96
480
Panel type
Cataloged
Cataloged
Custom
Custom
Custom
Cataloged
Cataloged and
Mix-n-Match
Custom
PCR Kit
required
181940
181942
181942
181942
181942
181940
181942
Quantity
181942
5
1
1
3
3
4
1
1
Storage
GeneRead DNAseq Panel Kits are shipped on dry ice and should be stored at
–30°C to –15°C upon arrival. When stored properly at –30°C to –15°C, all
reagents are stable for up to 6 months after delivery. GeneRead DNAseq Panel
PCR Kits are shipped on cold packs. For long-term storage, keep tubes at –30°C
to –15°C. If the entire volume will not be used at once, we recommend dividing
into aliquots and storing at –30°C to –15°C. Avoid repeated freezing and
thawing. If stored under these conditions, GeneRead DNAseq Panel PCR Kits
are stable for 6 months after receipt.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
7
Intended Use
GeneRead DNAseq Targeted Panels and GeneRead DNAseq Panel PCR Kits
are intended for molecular biology applications. These products are not
intended for the diagnosis, prevention or treatment of a disease.
All due care and attention should be exercised in the handling of the products.
We recommend all users of QIAGEN products to adhere to the NIH guidelines
that have been developed for recombinant DNA experiments, or to other
applicable guidelines.
Safety Information
When working with chemicals, always wear a suitable lab coat, disposable
gloves and protective goggles. For more information, please consult the
appropriate safety data sheets (SDSs). These are available online in convenient
and compact PDF format at www.qiagen.com/safety where you can find, view
and print the SDS for each QIAGEN kit and kit component.
Quality Control
GeneRead DNAseq Panel Kits are tested, and each assay in the GeneRead
DNAseq Targeted Panels is tested when designed, against predetermined
specifications to ensure consistent product quality.
8
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Introduction
DNA sequencing is a useful tool to detect genetic variations, including somatic
mutations, SNPs and small insertions and deletions. Targeted enrichment
technology enables next-generation sequencing (NGS) platform users to
sequence specific regions of interest instead of the entire genome, effectively
increasing sequencing depth and throughput with lower cost. GeneRead
DNAseq Targeted Panels V2 use multiplex PCR-based targeted enrichment
technology, in combination with a sophisticated primer design algorithm, to
enable amplification and enrichment of any gene or targeted region in the
human genome in order to detect genetic variation using NGS (Figure 1).
Adjacent and potentially interacting primer pairs are separated into different
pools for optimal performance. GeneRead DNAseq Targeted Panels V2 are
designed to analyze a panel of genes and/or regions related to a disease state
and can be used with any major NGS platform. The targeted enrichment
process is essential for the efficient utilization of medium- and high-throughput
sequencers such as Life Technologies®, Ion Personal Genome Machine®
(PGM™) Sequencer and Ion Proton™, as well as Illumina®’s MiSeq® Personal
Sequencer, HiSeq® 1000, HiSeq 1500, HiSeq 2000, HiSeq 2500 and GAIIx.
GeneRead DNAseq Targeted Panels V2 have been optimized in combination
with GeneRead DNAseq Panel PCR Kits V2 to provide superior sensitivity and
linear multiplex amplification. The simplicity of the PCR method makes these
panels accessible for routine use in every research laboratory.
Principle and procedure
GeneRead DNAseq Targeted Panels V2 are provided as sets of 1, 2, 3 or 4
pools, each containing primer mixes, with up to 9600 primer pairs per 4-pool
set. The number of pools included is determined by the region covered by
amplicons. Most panels cover full coding regions of genes, so one 4-pool set is
provided. For special panels, like the Tumor Actionable Mutations Panel
covering tumor mutation hotspots, one pool is provided. GeneRead DNAseq
Targeted Panels V2 can enrich selected genes and/or regions using as little as
40 or 20 ng genomic DNA in 3 hours for a 4- or 1-pool panel, respectively
(Figure 2). Briefly, genomic DNA samples are combined with primer mix and
PCR reagent and PCR is performed in a standard thermocycler. After the
reaction is complete, the reactions for each sample are pooled and the enriched
DNA is purified. The purified DNA then is ready for NGS library construction
and sequencing using the NGS platform of your choice. Since amplicons from
each sample are pooled before library construction, each sample results in one
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
9
library only. The sequencing results can be analyzed using the GeneRead
DNAseq Analysis Software at http://ngsdataanalysis.sabiosciences.com, which
will automatically perform all steps necessary to generate a DNA sequence
variant report from your NGS data (Figure 3). Alternatively, sequencing results
can be analysed by the Biomedical Genomics Workbench platform. All
detected variants can be interpreted by the Ingenuity Variant Analysis tool
(https://www.qiagen.com/us/products/catalog/sample-technologies/dnasample-technologies/genomic-dna/generead-dnaseq-gene-panelsv2/#resources).
Figure 1. Multiplex PCR-based targeted enrichment scheme. GeneRead DNAseq Targeted
Panels V2 use multiplex PCR-based targeted enrichment technology in combination with a
sophisticated primer design algorithm to maximize design coverage and minimize nonspecific
amplification. The adjacent primer sets are distributed across an appropriate number of pools
to minimize nonspecific amplification products.
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GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Figure 2. The GeneRead DNAseq Targeted Panel V2 procedure.
Figure 3. Overview of the sample-to-insight NGS workflow with GeneRead DNAseq Targeted
Panels V2. The complete sample-to-insight procedure begins with DNA extraction, followed by
targeted enrichment with GeneRead DNAseq Targeted Panels V2, NGS library construction,
sequencing and data analysis using either the QIAGEN NGS Data Analysis Web Portal or
Biomedical Genomics Workbench. Follow-up experiments or sample verification against
specific targets can be performed with qBiomarker Somatic Mutation PCR Assays. Detected
variants can be interpreted with the Ingenuity® Variant Analysis tool.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
11
Equipment and Reagents to be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable
gloves, and protective goggles. For more information, consult the appropriate
safety data sheets (SDSs), available from the product supplier.
In addition to the GeneRead DNAseq Targeted Panel V2 and GeneRead
DNAseq Panel PCR Kit V2, the following supplies are required:
For genomic DNA isolation:
 See page 14 for specific recommendations.
For targeted enrichment:

High-quality, nuclease-free water. Do not use DEPC-treated water.

GeneRead DNA QuantiMIZE Array or Assay Kit if using FFPE samples
(QIAGEN cat. nos. 180642/180654)

Agencourt® AMPure® XP Kit (Beckman Coulter cat. no. A63880)

Microcentrifuge

1.5 ml LoBind tubes

0.2 ml PCR tubes, 96-well reaction plates, or PCR strips and caps

Thermal cycler

Multichannel pipettor

Single-channel pipettor

DNase-free pipet tips and tubes

0.2 ml 96-well PCR plate

QIAxcel Advanced with a QIAxcel DNA High Resolution Kit or Agilent®
2100 Bioanalyzer®

Agilent High Sensitivity DNA Kit (Agilent cat. no. 5067-4626)

DynaMag™-96 Side Magnet (Thermo Fisher cat no. 12331D)
For NGS library construction for Ion PGM and Proton Sequencers:

GeneRead DNA Library L Core Kit (QIAGEN cat. no. 180462)

GeneRead DNA L Amp Kit (QIAGEN cat. no. 180485)
12
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015

GeneRead Adapter L Set 1-plex (QIAGEN cat. no. 180922) or GeneRead
Adapter L Set 12-plex (QIAGEN cat. no. 180994)

Agencourt AMPure XP Kit (Beckman Coulter cat. no. A63880)

GeneRead DNAseq Library Quant Kit for Ion PGM Sequencer (QIAGEN
cat. no. 180601)

0.2 ml 96-well PCR plate

80% ethanol

96-well Thermal cycler

A real-time thermal cycler compatible with 96-well/100-well/384-well
plates

DynaMag-96 Side Magnet (Thermo Fisher cat no. 12331D)
For NGS library construction for Illumina MiSeq/HiSeq:

GeneRead DNA Library I Core Kit (12) (QIAGEN cat. no. 180432) or
GeneRead DNA Library I Core Kit (48) (QIAGEN cat. no. 180434)

GeneRead DNA I Amp Kit (QIAGEN cat. no. 180455)

GeneRead Adapter I Set A 12-plex (QIAGEN cat. no. 180985) or
GeneRead Adapter I Set B 12-plex (QIAGEN cat. no. 180986) for up to
12 samples, or both kits for up to 24 samples.

GeneRead DNAseq Library Quant Kit for Illumina (QIAGEN cat. no.
180601)

Agencourt AMPure XP Kit (Beckman Coulter cat. no. A63880)

0.2 ml 96-well PCR plate

80% ethanol

Thermal cycler

A real-time PCR machine compatible with 96-well/100-well/384-well
plates

DynaMag-96 Side Magnet (Thermo Fisher cat no. 12331D)
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
13
Important Notes
DNA preparation and quality control
High-quality DNA is essential for obtaining good sequencing results
The most important prerequisite for DNA sequence analysis is consistent, highquality DNA from every experimental sample. Therefore, sample handling and
DNA isolation procedures are critical to the success of the experiment. Residual
traces of proteins, salts, or other contaminants may either degrade the DNA or
decrease the efficiency of, if not block completely, the enzyme activities
necessary for optimal targeted genome amplification and real-time PCR
performance.
Recommended genomic DNA preparation method
The QIAGEN QIAamp® DNA Mini Kit (cat. no. 51304), QIAamp DNA FFPE
Tissue Kit (cat. no. 56404) and GeneRead DNA FFPE Kit (cat. no. 180134) are
highly recommended for the preparation of genomic DNA samples from fresh
tissues and FFPE tissue samples. Ensure that samples have been treated for the
removal of RNA, as RNA contamination will cause inaccuracies in DNA
concentration measurements. Do not omit the recommended RNase treatment
step to remove RNA. If genomic DNA samples need to be harvested from
biological samples for which kits are not available, please contact Technical
Support representatives for suggestions.
For best results, all DNA samples should be resuspended in DNase-free water or
alternatively in DNase-free 10 mM Tris buffer pH 8.0. Do not use DEPC-treated
water.
DNA quantification and quality control
For best results, all DNA samples should also demonstrate consistent quality
according to the following criteria:
Concentration and purity determined by UV spectrophotometry
The concentration and purity of DNA should be determined by measuring the
absorbance in a spectrophotometer. Prepare dilutions and measure absorbance
14
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
in 10 mM Tris·Cl,* pH 8.0. The spectral properties of nucleic acids are highly
dependent on pH.
A260:A280 ratio should be greater than 1.8
Concentration determined by A260 should be >2.5 µg/ml DNA.
DNA integrity
For best results, the genomic DNA should be greater than 2 kb in length with
some fragments greater than 10 kb. This can be checked by running a fraction
of each DNA sample on a 1% agarose gel.
FFPE DNA
If FFPE DNA will be used for GeneRead DNAseq Targeted Panels, the QIAGEN
GeneRead DNA QuantiMIZE Array or Assay Kit is recommended for
determining optimal DNA amount and PCR cycling conditions for each FFPE
DNA sample (see Appendix C).
* When working with chemicals, always wear a suitable lab coat, disposable gloves and protective
goggles. For more information, please consult the appropriate safety data sheets (SDSs), available from
the product supplier.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
15
Protocol: PCR Setup
Procedure
1. Dilute DNA to 2.5 ng/µl with DNase-free water in a LoBind tube. For each
sample, 20 ng (8 µl, 2.5 ng/µl) DNA is required for the 1-pool or 2-pool
panels, 30 ng (12 µl, 2.5 ng/µl) for the 3-pool panels, or 40 ng (16 µl, 2.5
ng/µl) for the 4-pool panels.
Note: Dilution of FFPE DNA samples should be determined by the QIAGEN
GeneRead DNA QuantiMIZE Array or Assay Kit for optimal results.
2. Determine the number of reactions needed. For a 1-pool panel, one 40 µl
reaction for each sample is required. For 2-, 3- or 4-pool panels, 2, 3, or 4
x 20 µl reactions for each sample are required. Prepare PCR strips or a PCR
plate according to the number of reactions. Label with sample names and
pool numbers.
3. Aliquot 8 µl (1-pool panel) or 4 µl (2-, 3- or 4-pool panels) of each DNA
sample into each PCR reaction. Figure 4 shows an example of a 4-pool
panel being set up for 3 samples.
Note: If the GeneRead DNA QuantiMIZE Array or Assay Kit is used, the
same volume of FFPE DNA will be used after diluting DNA samples
according to the GeneRead DNA QuantiMIZE Array or Assay Kit.
Figure 4. An example of how a 4-pool panel can be set up for 3 samples.
16
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
4. Prepare the PCR mix on ice according to Table 1 (1-pool panel) or Table 2
(2, 3 or 4-pool panel), and as shown in Figure 5. For each sample, 1, 2, 3
or 4 PCR mixes will be needed. Mix gently by pipetting up and down.
Table 1. Preparation of PCR mix for each primer mix pool (1-pool panel e.g.,
Tumor Actionable Mutations Panel)
Component
Per 1 sample (µl)
Per n samples (µl)
GeneRead DNAseq Panel
PCR Buffer (5x)
8.8
8.8 x n
Primer mix pool (2x)
22
22 x n
GeneRead HotStarTaq
DNA Polymerase (6 U/µl)
2.9
2.9 x n
DNase-free water
1.5
1.5 x n
Total volume
35.2
35.2 x n
Table 2. Preparation of PCR mix for each primer mix pool (2, 3 or 4-pool panels)
Component
Per 1 sample (µl)
Per n samples (µl)
GeneRead DNAseq Panel
PCR Buffer (5x)
4.4
4.4 x n
Primer mix pool x* (2x)
11
11 x n
GeneRead HotStarTaq
DNA Polymerase (6 U/µl)
1.5
1.5 x n
DNase-free water
0.7
0.7 x n
Total volume
17.6
17.6 x n
* The number of primer mix pools is determined by the panel size. Primer pools are named A1
through A4.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
17
Figure 5. Preparation of PCR mastermix for each primer mix pool. For ease of setup, prepare
enough PCR mastermix to support all the primer pools and samples (left). Then, prepare poolspecific mastermixes (right, MM A1, MM A2 …).
5. Aliquot 32 µl (1-pool panel) or 16 µl (2, 3 or 4-pool panel) of each PCR
mix, and add it to the well with DNA samples accordingly, as shown in
Figure 6. Mix gently by pipetting up and down.
18
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Figure 6. Example of a 4-pool panel. DNA samples have already been aliquoted into wells in
step 3. Do not add anymore DNA.
6. Seal the wells with PCR tube caps. Place strips or plate in thermocycler and
set up reaction parameters according to Tables 3, 4A and 4B.
Table 3. PCR program
Cycle
Temperature
Time
1
95°C
15 min
Number of cycles
according to tables 4A
or 4B
95°C
15 s
60°C
4/8 min*
1
72°C
10 min
1
4°C
∞
* If number of primer pairs is <1200 in each pool, 4 min; if number of primer pairs is 1201–
2500 per pool, 8 min. To determine number of primer pairs per pool, refer to tables 4A or
4B.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
19
Table 4A. PCR cycles (if using a cataloged panel, refer to Table 4B)
*
Primer pairs per pool
No. of cycles for standard DNA*
1–11
25
12–23
24
24–47
23
48–95
22
96–191
21
192–287
20
288–399
19
400–1200
18
1201–2500
16
Number of cycles for FFPE samples should be determined by QIAGEN GeneRead DNA
QuantiMIZE Array or Assay Kit.
20
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Table 4B. PCR cycles and cycling times for cataloged panels
Cat #
NGHS001X
NGHS002X
NGHS003X
NGHS004X
NGHS005X
NGHS006X
NGHS007X
NGHS008X
NGHS009X
NGHS011X
NGHS013X
NGHS501X
NGHS101X
Panel name
# primer
pairs
# pools
# primer
pairs per
pool
# standard
PCR cycles
needed
Cycling
time (min)
Breast cancer
2915
4
729
18
4
1954
4
489
18
4
2536
4
634
18
4
Liver cancer
2052
4
513
18
4
Lung cancer
3586
4
897
18
4
Ovarian cancer
2021
4
506
18
4
Prostate cancer
1837
4
460
18
4
Gastric cancer
2377
4
595
18
4
Cardiomyopathy
2657
4
665
18
4
Carrier testing
6943
4
1736
16
8
6582
4
1646
16
8
7951
4
1988
16
8
602
4
151
21
4
Colorectal
cancer
Myeloid
Neoplasms
Cancer
predisposition
Comprehensive
cancer
Clinically
relevant
NGHS102X
BRCA1 and
BRCA2
250
4
63
22
4
NGHS201X
Actionable
mutations
118
1
118
21
4
7. After the reaction is complete, place on ice and proceed with sample
pooling and purification using AMPure XP beads.
Note: If the samples are to be stored prior to purification, transfer them to a
–20°C freezer. Samples are stable for 3 days.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
21
Protocol: Sample Pooling and Purification
1. For a 2, 3 or 4-pool panel, combine all 2, 3 or 4 reactions from the same
sample into one well of a PCR plate/strip. Mix thoroughly. The volume of
each sample should be approximately 40 µl for a 1- or 2-pool panel and
60 or 80 µl for a 3- or 4-pool panel.
2. Transfer 40 µl from each sample to a 1.5 ml LoBind tube or 96-well PCR
plate for purification.
3. Add 36 µl (0.9x volume) AMPure XP beads to 40 µl PCR product. Mix well
by pipetting.
4. Incubate for 5 min at room temperature.
5. Place the tube or 96-well PCR plate on magnetic rack to separate beads
from supernatant. After the solution is clear (approximately 5 min), carefully
transfer 70 µl of the supernatant to a new tube or wells in 96-well plate
without disturbing the beads. Discard the beads, which contain unwanted
large DNA fragments.
Note: Do not discard the supernatant.
IMPORTANT: Transferring 70 µl supernatant will leave behind about 6 µl
supernatant. This is to ensure that no beads are carried over into the
supernatant. Any bead carryover will result in a significant amount of larger
fragments present in the library, which will affect sequencing specificity.
6. Add 64 µl (1.6x the original volume of PCR product, which was 40 µl)
AMPure XP beads to the supernatant, mix well by pipetting and incubate for
5 min at room temperature.
7. Place the tube or 96-well PCR plate on a magnetic rack and wait until
solution is clear (approximately 5 min). Carefully remove and discard
supernatant. Be careful not to disturb the beads, which contain the DNA
target.
Note: Do not discard the beads.
8. Add 200 µl fresh 80% ethanol to the tube or well while it is on the magnetic
rack. Rotate the tube or move the plate side-to-side in the two positions of
the magnet to wash the beads, then carefully remove and discard the
supernatant.
9. Repeat previous step once.
10. Completely remove ethanol and dry beads for 15 min while the tube or
plate is on the rack.
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GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
11. Elute DNA target beads in 28 µl nuclease-free water. Mix well by pipetting.
Place tube or plate on magnetic rack until solution is clear. Transfer 25 µl
supernatant to a clean LoBind tube or well in the PCR plate.
12. Determine the amount of your sample with a QIAxcel Advanced with a
QIAxcel DNA High Resolution Kit or an Agilent 2100 Bioanalyzer, using
the High Sensitivity DNA Kit. Normally 15–60 ng of PCR product will be
obtained after purification.
13. Proceed to library construction according to the sequencing platform of your
choice. Refer to Appendix A for the recommended library construction
protocol for sequencing with Illumina MiSeq/HiSeq. Refer to Appendix B for
the recommended library construction protocol for sequencing with Ion PGM
Sequencer.
IMPORTANT: The protocols described in Appendix A and B are different
from the protocols described in the GeneRead Library Prep (I) Handbook.
For preparing libraries using panels, follow the steps described here only.
Note: If reactions are to be stored prior to library construction, transfer them
to a –20°C freezer. Samples are stable for 3 days.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
23
Troubleshooting Guide
For technical support, please call us at 1-888-503-3187 or 1-301-682-9200.
For more information, see also the Frequently Asked Questions page at our
Technical Support Center: www.qiagen.com/FAQ/FAQList.aspx. The
scientists in QIAGEN Technical Services are always happy to answer any
questions you may have about either the information and protocols in this
handbook or sample and assay technologies (for contact information, see back
cover or visit www.qiagen.com).
References
QIAGEN maintains a large, up-to-date online database of scientific publications
utilizing QIAGEN products. Comprehensive search options allow you to find the
articles you need, either by a simple keyword search or by specifying the
application, research area, title, etc.
For a complete list of references, visit the QIAGEN Reference Database online
at www.qiagen.com/RefDB/search.asp or contact QIAGEN Technical
Services or your local distributor.
24
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Appendix A: Library Construction Using GeneRead
Library Prep Kits for Illumina
The following kits are required for NGS library construction for Illumina
MiSeq/HiSeq:

GeneRead DNA Library I Core Kit (QIAGEN cat. no. 180432 or 180434)

GeneRead DNA I Amp Kit (QIAGEN cat. no. 180455)

GeneRead Adapter I Set A 12-plex (QIAGEN cat. no. 180985) or
GeneRead Adapter I Set B 12-plex (QIAGEN cat. no. 180986) for up to
12 samples, or both kits for up to 24 samples.

Agencourt AMPure XP Kit (Beckman Coulter cat. no. A63880)

GeneRead DNAseq Library Quant Kit for Illumina (QIAGEN cat. no.
180601)
End repair of DNA
Note: 10–200 ng PCR-enriched DNA should be used for library construction.
Starting amounts <10 ng or >200 ng will decrease the efficiency of library
construction.
A1.
Prepare a reaction mix for end-repair according to Table 5, dispensing
the reagents into a PCR tube or the well of a 96-well PCR plate on ice.
Table 5. Reaction mix for end-repair
Component
Volume (µl)
PCR-enriched DNA from previous step
20.5
End-Repair Buffer, 10x
2.5
End-Repair Enzyme Mix
2.0
Total
25
A2.
Mix the components by pipetting up and down several times.
A3.
Incubate in a thermal cycler for 30 min at 25°C, followed by
20 min at 75°C.
A4.
Pulse-spin the microfuge tube and return to ice.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
25
Note: If reactions are to be stored during library construction, transfer them to a
–20°C freezer. Samples are stable for 3 days.
A-addition
A5.
Prepare a reaction mix for A-addition according to Table 6, adding the
components to the PCR tube or plate containing the end-repaired DNA
from step A4.
Table 6. Reaction mix for A-addition
Component
Volume
End-repaired DNA (from step A4)
25 µl
A-addition Buffer, 10x
3 µl
Klenow Fragment (3’5’ exo-)
3 µl
Total
31 µl
A6.
Mix the components by pipetting up and down several times.
A7.
Incubate in a thermal cycler for 30 min at 37°C, followed by
10 min at 75°C.
Note: If reactions are to be stored during library construction, transfer them to a
–20°C freezer. Samples are stable for 3 days.
Adapter ligation
A8.
Prepare a reaction mix for adapter ligation according to Table 7, adding
the components to the PCR tube or plate containing DNA that has
undergone end-repair and A-addition (step A7).
Note: When using barcode adapters, open one adapter tube at a time
and change gloves between pipetting the different barcode adapters to
avoid cross-contamination.
IMPORTANT: Only one of the 24 adapters (Adapter Bc1–Bc127) should
be used per ligation reaction.
26
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Table 7. Reagents for adapter ligation of PCR product
Component
Volume/reaction (µl)
DNA from step A7
31
Ligation Buffer, 2x
45
Adapter†
1*
T4 DNA Ligase
4
DNase-free water
9
Total
90
* If other suppliers’ adapters will be used, use 0.28 µM final concentration or add the correct
amount of adapter according to supplier’s directions. If more than 24 samples will be
multiplexed, NEXTflex™ DNA Barcodes from Bioo Scientific can be used.
† This applies to GeneRead Adapter I Set A 12-plex and GeneRead Adapter I Set B 12-plex
A9.
Mix the components by pipetting up and down several times.
A10. Program a thermocycler to incubate at 25°C for 10 min.
IMPORTANT: Do not use a thermocycler with a heated lid.
A11. After the reaction is complete, place the reactions on ice and proceed
with purification using AMPure XP beads.
Note: If reactions are to be stored during library construction, transfer them to a
–20°C freezer. Samples are stable for 3 days.
Cleanup of adapter-ligated DNA with AMPure XP beads
A12. Transfer 90 µl ligation reaction from A11 to a 1.5 ml LoBind tube if using
tube, keep ligation reaction in PCR plate if using plate protocol.
A13. Add 108 µl (1.2 x volume) AMPure XP beads to 90 µl DNA solution. Mix
well by pipetting up and down several times.
A14. Incubate for 5 min at room temperature.
A15. Place the tube or 96-well PCR plate on magnetic rack to separate beads
from supernatant. After the solution is clear (approximately 5–10 min),
carefully remove and discard supernatant. Be careful not to disturb the
beads, which contain the DNA target.
Note: Do not discard the beads.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
27
A16. Add 200 µl freshly made 80% ethanol to the tube or plate while it is on
the magnetic rack. Rotate the tube or move the plate side-to-side in the
two positions of the magnet to wash the beads. Carefully remove and
discard the supernatant.
A17. Repeat previous step once.
A18. Completely remove ethanol and dry beads for 10 min while the tube or
plate is on the rack.
A19. Elute DNA target beads in 19 µl nuclease-free water. Mix well by
pipetting. Place tube or plate on the rack until solution is clear.
A20. Transfer 17 µl supernatant to a clean PCR tube or well in PCR plate and
proceed to PCR amplification.
Note: The median size of the library will be 280 bp.
Note: If reactions are to be stored after library construction, transfer them
to a –20°C freezer. Samples are stable for 3 days.
PCR amplification of purified library
A21. Mix the components in Table 8 in a 0.2 ml PCR tube or 96-well PCR
plate.
Table 8. Reaction components for PCR amplification
Component
Volume/reaction (µl)
HiFi PCR Master Mix, 2x
25
Primer Mix (10 µM each)*
1.5
Library DNA (from step A20)
17
RNase-free water
6.5
Total
50
* Use 0.3 µM final concentration of the PCR primer mix. Alternatively, add the correct amount
of primer according to supplier’s directions.
A22. Set up the cycler using the cycling conditions in Table 9.
28
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Table 9. Cycling conditions for amplification of the DNA library
Step
Temperature
Time
Initial denaturation
98°C
2 min
4 cycles
98°C
20 sec
60°C
30 sec
72°C
30 sec
72°C
1 min
4°C
∞
1 cycle
Hold
A23. After the reaction is complete, place the reactions on ice and proceed
with cleanup with AMPure XP beads.
Note: If reactions are to be stored after library amplification, transfer them to a
–20°C freezer. Samples are stable for 3 days.
Cleanup of amplified library with AMPure XP beads
A24. Transfer 50 µl PCR reaction from A23 to a 1.5 ml LoBind tube if using
tube, keep PCR reaction in PCR plate if using plate protocol.
A25. Add 40 µl (0.8x volume) AMPure XP beads to 50 µl PCR solution. Mix
well by pipetting up and down several times.
A26. Incubate for 5 min at room temperature.
A27. Place the tube or 96-well PCR plate on magnetic rack to separate beads
from supernatant. After the solution is clear (approximately 5 min),
carefully transfer 86 µl of the supernatant to a new tube or well in 96-well
plate without disturbing the beads. Discard the beads, which contain
unwanted large DNA fragments.
Note: Do not discard the supernatant.
A28. Add 20 µl (0.4 x the original volume of PCR product, which was 50 µl)
AMPure XP beads to the supernatant, mix well by pipetting and incubate
for 5 min at room temperature.
A29. Place the tube or 96-well PCR plate on a magnetic rack and wait until
solution is clear (approximately 5 min). Carefully remove and discard
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
29
supernatant. Be careful not to disturb the beads, which contain the DNA
target.
Note: Do not discard the beads.
A30. Add 200 µl fresh 80% ethanol to the tube or well while it is on the
magnetic rack. Rotate the tube or move the plate side-to-side in the two
positions of the magnet to wash the beads, then carefully remove and
discard the supernatant.
A31. Repeat previous step once.
A32. Completely remove ethanol and dry beads for 10 min while the tube or
plate is on the rack.
A33. Elute DNA library beads in 30 µl nuclease-free water or appropriate
buffer. Mix well by pipetting. Place tube or plate on magnetic rack until
solution is clear. Transfer 28 µl supernatant to a clean LoBind 1.5 ml tube
or PCR tube.
A34. The library can be stored in a –20°C freezer prior to quantification using
the GeneRead DNAseq Library Quant Array. Amplified libraries are
stable for 1 month at –20°C.
30
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Appendix B: Library Construction Using GeneRead
Library Prep Kits for Ion PGM Sequencer/Proton
The following kits are required for NGS library construction for the Ion PGM
Sequencer or Ion Proton:
 GeneRead DNA Library L Core Kit (QIAGEN cat. no. 180462)

GeneRead DNA L Amp Kit (QIAGEN cat. no. 180485)

GeneRead Adapter L Set 1-plex (QIAGEN cat. no. 180922) or GeneRead
Adapter L Set 12-plex (QIAGEN cat. no. 180994)

Agencourt AMPure XP Kit (Beckman Coulter cat. no. A63880)

GeneRead DNAseq Library Quant Kit for Ion PGM Sequencer (QIAGEN
cat. no. 180601)
End repair of DNA
Note: 10–200 ng PCR-enriched DNA should be used for library construction.
Starting amounts <10 ng or >200 ng will decrease the efficiency of library
construction.
B1.
Prepare a reaction mix for end-repair according to Table 10, dispensing
the reagents into a PCR tube or the wells of a PCR plate on ice.
Table 10. DNA end-repair reaction components
Component
Volume (µl)
PCR-enriched DNA from previous step
20.5
End-Repair Buffer, 10x
2.5
End-Repair Enzyme Mix
2.0
Total
25
B2.
Mix the components by pipetting up and down several times.
B3.
Incubate in a thermal cycler for 20 min at 25°C, followed by
10 min at 70°C.
B4.
Pulse-spin the microfuge tube and return to ice.
Note: If reactions are to be stored during library construction, transfer
them to a –20°C freezer. Samples are stable for 3 days.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
31
Adapter ligation
B5.
Prepare a reaction mix for adapter ligation according to Table 11,
adding the components to the PCR tube containing the end-repaired DNA
from previous step. Mix thoroughly.
Note: If analyzing 1 sample only, use the GeneRead Adapter L Set
1-plex. If analyzing up to 12 samples in a single run, use the GeneRead
Adapter L Set 12-plex. If analyzing more than 12 samples, refer to table
11 for instructions.
Note: When using barcode adapters, open one adapter tube at a time
and change gloves between pipetting the different barcode adapters to
avoid cross-contamination.
IMPORTANT: Only one of the 12 adapters (Adapter Bc1–Bc12) should be
used per ligation reaction, in combination with the universal adapter
BcGen.
32
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Table 11. Reaction setup for adapter ligation
Component
Singleplex
Volume/reaction (µl)
Multiplex
Volume/reaction (µl)
End-repaired DNA (from step
B4)
25
25
Ligation Buffer, 2x
40
40
0.5*

Universal Adapter BcGen

0.5†
Barcode Adapter 1–12

0.5†
Ligation and Nick Repair Mix
4
4
dNTP Mix (10 mM)
1
1
DNase-free water
9.5
9
Total
80.0
80.0
Adapter (singleplex)
*
If other suppliers’ adapter will be used, use 0.16 µM final concentration or add the correct
amount of adapter according to supplier’s directions. † If more than 12 samples will be
multiplexed, Ion Xpress™ Barcode Adapters from Life Technologies can be used. For each
reaction, use 0.5 µl Ion Xpress P1 Adapter, and 0.5 µl of one of the Ion Xpress Barcodes.
Only one of the Ion Xpress Barcodes is used for each sample.
B6.
Mix the contents by pipetting up and down several times.
B7.
Program a thermocycler to incubate for 10 min at 25°C, followed by 5
min at 72°C.
IMPORTANT: Do not use a thermocycler with a heated lid.
B8.
After the reaction is complete, place the reactions on ice and proceed
with purification using AMPure XP beads.
Note: If reactions are to be stored during library construction, transfer
them to a –20°C freezer. Samples are stable for 3 days.
Cleanup of adapter-ligated DNA with AMPure XP beads
B9.
Transfer 80 µl ligation reaction from B8 to a 1.5 ml LoBind tube if using
tube, keep ligation reaction in PCR plate if using plate protocol.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
33
B10. Add 112 µl (1.4x volume) AMPure XP beads to 80 µl DNA solution. Mix
well by pipetting up and down several times.
B11. Incubate for 5 min at room temperature.
B12. Place the tube or 96-well PCR plate on magnetic rack to separate beads
from supernatant. After the solution is clear (approximately 5–10 min),
carefully remove and discard supernatant. Be careful not to disturb the
beads, which contain the DNA target.
Note: Do not discard the beads.
B13. Add 200 µl freshly made 80% ethanol to the tube or plate while it is on
the magnetic rack. Rotate the tube or move the plate side-to-side in the
two positions of the magnet to wash the beads. Carefully remove and
discard the supernatant.
B14. Repeat previous step once.
B15. Completely remove ethanol and dry beads for 10 min while the tube or
plate is on the rack.
B16. Elute DNA target beads in 19 µl nuclease-free water. Mix well by
pipetting. Place tube or plate on the rack until solution is clear.
B17. Transfer 17 µl supernatant to a clean PCR tube or well in PCR plate and
proceed to PCR amplification.
Note: The median size of the library will be 220 bp.
Note: If reactions are to be stored after library construction, transfer them
to a –20°C freezer. Samples are stable for 3 days.
PCR amplification of the purified library
Note: This step is required to ensure that only properly-made libraries proceed
to the next-generation sequencing step.
B18. Mix the components in Table 12 in a 0.2 ml PCR tube or plate.
34
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Table 12. Reaction components for PCR amplification
Component
Volume (µl)
HiFi PCR Master Mix, 2x
25
Primer Mix (10 µM each)
1.5
Library DNA (from step B17)
17
RNase-free water
6.5
Total
50
B19. Set up the cycler using the cycling conditions in Table 13.
Table 13. Cycling conditions for amplification of adapter-ligated DNA
Step
Temperature
Time
Initial denaturation
98°C
2 min
5 cycles
98°C
20 sec
60°C
30 sec
72°C
30 sec
72°C
1 min
4°C
∞
1 cycle
Hold
B20. After the reaction is complete, place the reactions on ice and proceed
with cleanup using the AMPure XP beads.
Note: If reactions are to be stored during library amplification, transfer
them to a –20°C freezer. Samples are stable for 3 days.
Cleanup of amplified library with AMPure XP beads
B21. Transfer 50 µl PCR reaction from B20 to a 1.5 ml LoBind tube if using
tube, keep reaction in PCR plate if using plate protocol.
B22. Add 70 µl (1.4 x volume) AMPure XP beads to 50 µl PCR reaction. Mix
well by pipetting up and down several times.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
35
B23. Incubate for 5 min at room temperature.
B24. Place the tube or 96-well PCR plate on magnetic rack to separate beads
from supernatant. After the solution is clear (approximately 5 min),
carefully remove and discard supernatant. Be careful not to disturb the
beads, which contain the DNA target.
Note: Do not discard the beads.
B25. Add 200 µl freshly made 80% ethanol to the tube or plate while it is on
the magnetic rack. Rotate the tube or move the plate side-to-side in the
two positions of the magnet to wash the beads. Carefully remove and
discard the supernatant.
B26. Repeat previous step once.
B27. Completely remove ethanol and dry beads for 10 min while the tube or
plate is on the rack.
B28. Elute DNA library beads in 30 µl nuclease-free water or appropriate
buffer. Mix well by pipetting. Place tube or plate on magnetic rack until
solution is clear. Transfer 28 µl supernatant to a clean LoBind 1.5 ml tube
or PCR tube.
B29. The library can be stored in a –20°C freezer prior to quantification using
the GeneRead DNAseq Library Quant Array. Amplified libraries are
stable for 1 month at –20°C.
36
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Appendix C: FFPE DNA Quality and Quantity
Genomic DNA present in FFPE archives is usually damaged and fragmented to
an uncertain extent. Commonly used DNA quantification methods including
spectrometers or fluorometers do not differentiate between amplifiable and nonamplifiable DNA. Therefore, they cannot reliably measure the amplifiable
amounts of DNA that are able to participate in the multiplex PCR-based targeted
enrichment step in the NGS workflow.
The QIAGEN GeneRead DNA QuantiMIZE System is a qPCR-based approach
that determines the quantity and quality of DNA that is amenable to PCR-based
targeted enrichment prior to NGS. The system provides a sensitive and accurate
approach to qualify and quantify DNA isolated from biological samples, mainly
for FFPE samples. Please refer to the corresponding user manual for determining
FFPE DNA quantity and quality with GeneRead DNA QuantiMIZE System.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
37
Appendix D: Library Quantification and Quality Control
Quality control for the targeted enrichment and library construction process can
be performed using QIAGEN’s GeneRead DNAseq Library Quant Array (cat.
no. 180601). With this array, the correct dilution of the library can also be
determined for sequencing. Please refer to the corresponding user manual for
library quantification and quality control.
38
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Appendix E: Data Analysis using QIAGEN’s GeneRead
DNAseq Sequence Variant Analysis Software
After sequencing, results can be analyzed using QIAGEN’s Cloud-Based
GeneRead DNAseq Sequence Variant Analysis Software. Our data analysis
software will perform mapping to the reference genome, read trimming
(removing primer sequences) and variant identification. Please refer to the
corresponding document for data analysis. Alternatively, sequencing results can
be analysed by the Biomedical Genomics Workbench platform. All detected
variants can be interpreted by the Ingenuity Variant Analysis tool
(https://www.qiagen.com/us/products/catalog/sample-technologies/dnasample-technologies/genomic-dna/generead-dnaseq-gene-panelsv2/#resources).
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
39
Appendix F: Combine Libraries for Multiplex Sequencing
Libraries can be combined into one sequencing run, as long as each library
uses a different barcode.
Ion PGM Sequencer libraries
Barcoded libraries can be constructed using the GeneRead Adapter L Set 12plex. If more than 12 samples will be multiplexed, the Ion Xpress Barcode
Adapters from Life Technologies (cat. nos. 4471250, 4474009, 4474518,
4474519, 4474520, 4474521, or 4474517) can be used to replace the
“GeneRead Adapter L Set 12-plex” in Table 11, page 33.
After the library is constructed, follow Appendix D to determine the library
dilution factor (which dilutes libraries to 4–8 pM) and dilute each individual
library according to this factor. Combine libraries in equimolar amounts, and
mix well. At least 25 µl of the mixture is required. Proceed to template
preparation using the mixture.
Illumina libraries
Barcoded libraries can be constructed using GeneRead Adapter I Set A 12-plex
or GeneRead Adapter I Set B 12-plex as described in Appendix A, Table 7. If
more than 24 samples will be multiplexed, replace the GeneRead Adapters with
NEXTflex™ DNA Barcodes from Bio Scientific, as described in Appendix A,
Table 7.
After the library is constructed, follow Appendix D to determine the library
concentration. Dilute individual libraries to 4 nM, then combine libraries in
equimolar amounts, and mix well. At least 5 µl of the mixture is needed.
Proceed to denature libraries using fresh NaOH and generate clusters using this
mixture.
40
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Appendix G: Analyze the PCR amplicons and Library
Using the QIAxcel Advanced with a QIAxcel DNA High
Resolution Kit or Agilent 2100 Bioanalyzer
The QIAxcel Advanced with a QIAxcel DNA High Resolution Kit or Agilent
2100 Bioanalyzer can be used at multiple steps of the targeted enrichment
workflow as a quality control checkpoint. If the Agilent 2100 Bioanalyzer is
used, the High Sensitivity DNA kit must be used.
After the multiplex PCR run, sample pooling, and purification, the PCR product
can be analyzed using the QIAxcel Advanced or an Agilent 2100 Bioanalyzer.
A sample image from both instruments is shown in Figure 7. The amplicons
should be in the correct size range (usually around 160 bp), and the measured
amount of DNA under the appropriate peak should be greater than 10 ng total.
The OD reading (NanoDrop®) method is not recommended for determination of
DNA concentration because the product may be below the detection limitation
of the instrument.
Figure 7. QIAxcel (top) and Bioanalyzer (bottom) trace of pooled and purified multiplex PCR
product. A peak of around 160 bp is observed, which represents the amplicons. The primer
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
41
dimers will be removed during purification steps after adapter ligation and library
amplification and, therefore, will have no effect on the final library (see Figure 8).
After the library is constructed, amplified and purified, the QIAxcel Advanced or
Bioanalyzer can be used to check the fragment size and concentration. For Ion
PGM Sequencer libraries, a peak around 220 bp is expected. There should be
no significant peak at around 100 bp, which represents adapter dimers. For
Illumina libraries, a peak around 280 bp is expected (Figure 8) and no
significant peak should be observed around 120 bp, which represents adapter
dimers. Amounts of DNA under the appropriate peaks can be used to quantify
libraries. However, due to the superior sensitivity of qPCR, we recommend
quantifying libraries with the GeneRead DNAseq Library quantification kits (cat.
no. 180601).
Figure 8. Sample Agilent Bioanalyzer image of a MiSeq Sequencer library. A peak of around
280 bp is observed.
42
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Appendix H: Downloading Individual Unaligned .BAM File
with a Multiplex Sample on Ion PGM Sequencer
H1.
When the run is finished, navigate to the report page on the Torrent
Browser. Locate the “Output Files” section near the end of the report.
Figure 9. Report page and output files.
H2.
Click the “UBAM” button in the row corresponding to individual
barcoded samples and column labeled “Files” in the table. These are the
unaligned reads in BAM format, with the barcode separated for each
sample. Save the .bam file to your local disk. The file is usually several
hundred megabytes to several gigabytes, depending on the size of the
sequencing chip being used.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
43
Figure 10. Unaligned reads in BAM format.
H3.
Locate the file that was just downloaded to the local disk and upload the
individual .BAM file to QIAGEN’s NGS Sequence Variant Analysis Web
Portal for analysis.
44
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Ordering Information
Product
Contents
Cat. no.
GeneRead DNAseq
Targeted Panels V2
Sets of 1 or 4 pools containing
wet-bench verified primer sets for
targeted enrichment of a focused
panel of <100 genes
181900
GeneRead DNAseq
Targeted HC Panel V2
Sets of 4 pools containing wetbench verified primer sets for
targeted enrichment of a focused
panel of >100 genes
181901
GeneRead DNAseq
Custom Panel V2
Pools containing primer sets for
targeted enrichment of a
customized panel of genes or
genomic regions
181902
GeneRead DNAseq
Mix-n-Match Panel V2
Pools containing wet-bench
verified primer sets for targeted
enrichment of a custom panel of
genes
181905
GeneRead DNAseq
Panel PCR Kit V2
PCR chemistry for use with the
GeneRead DNAseq Panel V2
System
Varies
Related products
GeneRead DNAseq
Library Quant Array
Reagents for NGS sample library
quantification following targeted
enrichment with the GeneRead
DNAseq Targeted Panel System
GeneRead qPCR
SYBR® Green
Mastermix
Mastermix for use with the
GeneRead Library Quant Arrays
and Kit
GeneRead DNA
QuantiMIZE Array Kit
qPCR arrays for optimizing amount
of input DNA and PCR cycling
conditions for targeted enrichment
of FFPE DNA
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
180601
Varies
180642
45
Product
Contents
Cat. no.
GeneRead DNA
QuantiMIZE Assay Kit
qPCR assays for optimizing
amount of input DNA and PCR
cycling conditions for targeted
enrichment of FFPE DNA
180654
QIAamp DNA Mini Kit For 50 DNA preps: 50 QIAamp
(50)
Mini Spin Columns, QIAGEN
Proteinase K, Collection Tubes (2
ml), reagents and buffers
51304
QIAamp DNA FFPE
Tissue Kit (50)
For 50 DNA preps: 50 QIAamp
MinElute® Columns, Proteinase K,
Collection Tubes (2 ml), buffers
56404
GeneRead DNA FFPE
Kit (50)
QIAamp MinElute columns,
Proteinase K, UNG, Collection
Tubes (2 ml), Buffers,
Deparaffinization Solution,
RNaseA
180134
Ion PGM Sequencer/Proton Library Prep
GeneRead DNA
Library L Core Kit (12)
For 12 reactions: Buffers and
reagents for end-repair, ligation,
and nick repair, for use with Ion
PGM Sequencer/Proton
Instruments from Life Technologies
180462
GeneRead DNA L
Amp Kit (100)
For 100 reactions: Buffers and
reagents for library amplification,
for use with Ion PGM
Sequencer/Proton Instruments from
Life Technologies
180485
GeneRead Adapter L
Set 12-plex (72)
For 72 reactions: 12 barcoded
adapters for ligation to DNA
library, for use with instruments
from Life Technologies
180994
GeneRead Adapter L
Set 1-plex (12)
For 12 reactions: Adapters for
DNA ligation, for use with Life
Technologies instruments
180922
46
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
Product
Contents
Cat. no.
GeneRead DNA
Library I Core Kit (48)
For 48 reactions: Buffers and
reagents for end-repair, A-Addition,
and ligation, for use with Illumina
instruments
180434
GeneRead DNA I
Amp Kit (100)
For 100 reactions: Buffers and
reagents for library amplification,
for use with Illumina instruments
180455
GeneRead Adapter I
Set A 12-plex (144)
For 144 reactions: 12 barcoded
adapters for ligation to DNA
library, for use with Illumina
instruments
180985
GeneRead Adapter I
Set B 12-plex (144)
For 144 reactions: 12 barcoded
adapters for ligation to DNA
library, for use with Illumina
instruments
180986
Illumina Library Prep
For up-to-date licensing information and product-specific disclaimers, see the
respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and
user manuals are available at www.qiagen.com or can be requested from
QIAGEN Technical Services or your local distributor.
GeneRead DNAseq Targeted Panels V2 Handbook 06/2015
47
Trademarks: QIAGEN®, QIAamp®, QIAquick®, QIAxcel®, GeneRead™, HotStarTaq®, Ingenuity®, MinElute® (QIAGEN Group); Agilent®,
Biaanalyzer® (Agilent Technologies, Inc.); AMPure®, Agencourt® (Beckman Coulter, Inc.); HiSeq®, MiSeq®, Illumina® (Illumina, Inc.); Ion Xpress™, Ion
Proton™, Personal Genome Machine®, PGM™, SYBR®, Life Technologies® (Life Technologies Corporation); NanoDrop® (NanoDrop Technologies,
Inc.); NEBNext®, (New England BioLabs, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such,
are not to be considered unprotected by law.
Limited License Agreement for GeneRead DNAseq Targeted Panels V2
Use of this product signifies the agreement of any purchaser or user of the product to the following terms:
1.
The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components
contained in the kit only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this
kit with any components not included within this kit except as described in the protocols provided with the product, this handbook, and
additional protocols available at www.qiagen.com. Some of these additional protocols have been provided by QIAGEN users for QIAGEN
users. These protocols have not been thoroughly tested or optimized by QIAGEN. QIAGEN neither guarantees them nor warrants that they do
not infringe the rights of third-parties.
2.
Other than expressly stated licenses, QIAGEN makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.
3.
This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.
4.
QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.
5.
The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited
above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court
costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit
and/or its components.
For updated license terms, see www.qiagen.com.
© 2015 QIAGEN, all rights reserved.
www.qiagen.com
Australia  techservice-au@qiagen.com
Austria  techservice-at@qiagen.com
Belgium  techservice-bnl@qiagen.com
Brazil  suportetecnico.brasil@qiagen.com
Canada  techservice-ca@qiagen.com
China  techservice-cn@qiagen.com
Denmark  techservice-nordic@qiagen.com
Finland  techservice-nordic@qiagen.com
France  techservice-fr@qiagen.com
Germany  techservice-de@qiagen.com
Hong Kong  techservice-hk@qiagen.com
India  techservice-india@qiagen.com
Ireland  techservice-uk@qiagen.com
Italy  techservice-it@qiagen.com
Japan  techservice-jp@qiagen.com
Korea (South)  techservice-kr@qiagen.com
Luxembourg  techservice-bnl@qiagen.com
Mexico  techservice-mx@qiagen.com
The Netherlands  techservice-bnl@qiagen.com
Norway  techservice-nordic@qiagen.com
Singapore  techservice-sg@qiagen.com
Sweden  techservice-nordic@qiagen.com
Switzerland  techservice-ch@qiagen.com
UK  techservice-uk@qiagen.com
USA  techservice-us@qiagen.com
1094879
06/2015
Sample & Assay Technologies
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