TruSeq Amplicon v2.0
BaseSpace App Guide
For Research Use Only. Not for use in diagnostic procedures.
Introduction
Workflow Diagram
Set Analysis Parameters
Analysis Methods
Analysis Output
Revision History
Technical Assistance
ILLUMINA PROPRIETARY
Document # 15055857 v02
January 2016
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© 2016 Illumina, Inc. All rights reserved.
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The BaseSpace® App, TruSeq Amplicon v2.0, analyzes DNA enriched for particular
target sequences. The app aligns amplicon reads against the reference specified in the
manifest file, and then performs variant analysis. Variants are called for the targeted
regions. Statistics reporting accumulates coverage statistics for each target and overall
metrics.
Compatible Libraries
See the BaseSpace support page for a list of library types that are compatible with the
TruSeq Amplicon v2.0 App.
Workflow Requirements
}
}
}
}
}
}
The minimum read length is 50 bases.
No minimum number of reads is required. However, use sufficient data for each
sample to support an appropriate depth of coverage for variant calling.
Only 1 manifest is used per analysis.
Paired-end sequencing data is required.
Sequenced samples have the same read lengths.
A maximum of 96 samples is possible per analysis.
Versions
The following components are used in the TruSeq Amplicon v2.0 App.
Software
Version
TruSeq Amplicon (BaseSpace Workflow)
2.0.0
Isis (Analysis Software)
2.6.21.7
SAMtools
1.2
Somatic Variant Caller
4.0.13.1
Starling (Isaac Variant Caller)
2.1.4.1
GATK (Variant Caller)
v1.6-23-gf0210b3
IONA (Illumina On-Node Annotation)
1.0.10.37
MarkDuplicates
1.0.1
mono
3.12.1
This app was validated with data from MiSeq and NextSeq systems.
Reference Genomes
}
Human, UCSC hg19
TruSeq Amplicon v2.0 BaseSpace App Guide
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Introduction
Introduction
}
}
}
The human reference genome is PAR-Masked, which means that the Y chromosome
sequence has the Pseudo Autosomal Regions (PAR) masked (set to N) to avoid
mismapping of reads in the duplicate regions of sex chromosomes.
Cow, UMD3.1
Mouse, mm9
Rat, rn4
NOTE
When using a custom manifest file, specify a reference genome in the Build ID column.
Reference genome names are case sensitive.
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Document # 15055857 v02
Workflow Diagram
Workflow Diagram
Figure 1 TruSeq Amplicon v2.0 App Workflow
TruSeq Amplicon v2.0 BaseSpace App Guide
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Set Analysis Parameters
1
In BaseSpace, click the Apps tab.
2
Click TruSeq Amplicon.
3
From the drop-down list, select version 2.0, and then click Launch to open the app.
4
In the Analysis Name field on the app input form, enter the analysis name.
By default, the analysis name includes the app name, followed by the date and time
that the analysis session starts.
5
From the Save Results To field, select the project that stores the app results.
6
From the Sample(s) field, browse to the sample you want to analyze and select the
checkbox. You can select multiple samples.
7
From the Targeted Amplicons field, select a panel of targeted amplicons
representative of the selected samples.
8
If you selected Custom Panel in the Targeted Amplicons drop-down list, upload a
custom manifest and select the manifest file from the Custom Manifest File field.
Upload a custom manifest as follows.
a
b
c
Navigate to your project in BaseSpace.
Click Import.
Follow the instructions to add the Custom Amplicon manifest file (*.txt) to the
project.
NOTE
Specify the reference genome in the header of the manifest file. See Reference Genomes
on page 3.
9
From the Variant Caller field, select a variant caller.
For tumor samples, use the Somatic Variant Caller.
10 If using the somatic variant caller, specify the Somatic Variant Caller Threshold
(percentage).
Set to 5 by default. Variants with a frequency below the specified threshold are not
reported in VCF files. Lower threshold values can result in false positive variants.
11 Set the Read Stitching option.
When enabled, reads that overlap > 10 bases between Read 1 and Read 2 are
combined to create a single (longer) read for alignment.
12 From the Annotation field, select a preferred gene and transcript annotation reference
database.
13 Click Continue.
The TruSeq Amplicon v2.0 App begins analysis of the sample.
When analysis is complete, the status of the app session is updated automatically
and an email is sent to notify you.
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Document # 15055857 v02
The TruSeq Amplicon v2.0 workflow evaluates short regions of amplified DNA, or
amplicons, for variants. Focused sequencing of amplicons enables high coverage of
particular regions across many samples.
Alignment
During the alignment step, the banded Smith-Waterman algorithm aligns clusters from
each sample against amplicon sequences specified in the manifest file.
The banded Smith-Waterman algorithm performs local sequence alignments to
determine similar regions between 2 sequences. Instead of comparing the total sequence,
the Smith-Waterman algorithm compares segments of all possible lengths. Local
alignments are useful for dissimilar sequences that are suspected to contain regions of
similarity within the larger sequence. This process allows alignment across small
amplicon targets, often less than 10 bp.
Each paired-end read is evaluated in terms of its alignment to the relevant probe
sequences for that read.
} Read 1 is evaluated against the reverse complement of the Downstream LocusSpecific Oligos (DLSO).
} Read 2 is evaluated against the Upstream Locus-Specific Oligos (ULSO).
} If the start of a read matches a probe sequence with no more than 1 mismatch, the
full length of the read is aligned against the amplicon target for that sequence.
Alignments that include more than 3 indels are filtered from alignment results. Filtered
alignments are written in alignment files as unaligned and are not used in variant
calling.
Variant Calling
Variant calling is performed with any of the following variant calling options.
} GATK
} Isaac Variant Caller
} Somatic Variant Caller
GATK
Developed by the Broad Institute, the Genome Analysis Toolkit (GATK) first calls raw
variants for each sample read. Then GATK analyzes the variants against known
variants, and applies a calibration procedure to compute a false discovery rate for each
variant. Variants are flagged as homozygous (1/1) or heterozygous (0/1) in the VCF file
sample column.
The GATK best practices were guidelines for the app; they are described here:
www.broadinstitute.org/gatk/guide/topic?name=best-practices.
For more information about GATK, see www.broadinstitute.org/gatk.
DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del
Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ, Kernytsky AM, Sivachenko AY,
Cibulskis K, Gabriel SB, Altshuler D, Daly MJ (2011) A framework for variation discovery and
genotyping using next-generation DNA sequencing data. Nat Genet. 43(5): 491-8.
TruSeq Amplicon v2.0 BaseSpace App Guide
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Analysis Methods
Analysis Methods
Starling (Isaac Variant Caller)
Starling identifies single nucleotide variants (SNVs) and small indels using the following
steps:
} Read filtering—Filters out reads failing quality checks.
} Indel calling—Identifies a set of possible indel candidates and realigns all reads
overlapping the candidates using a multiple sequence aligner.
} SNV calling—Computes the probability of each possible genotype given the aligned
read data and a prior distribution of variation in the genome.
} Indel genotypes—Calls indel genotypes and assigns probabilities.
} Variant call output—Generates output in a VCF file and a compressed genome
variant call (gVCF) file. See VCF File Format on page 12 and Genome VCF Files on
page 14.
Indel Candidates
Input reads are filtered by removing any of the following reads:
} Reads that failed base calling quality checks
} Paired-end reads not marked as a proper pair
} Reads with a mapping quality < 20
Indel Calling
The variant caller proceeds with candidate indel discovery and generates alternate read
alignments based on the candidate indels. As part of the realignment process, the variant
caller selects a representative alignment to be used for site genotype calling and depth
summarization by the SNV caller.
SNV Calling
The variant caller runs a series of filters on the set of filtered and realigned reads for
SNV calling without affecting indel calls. First, any contiguous trailing sequence of N
base calls is trimmed from the ends of reads. Using a mismatch density filter, reads
having an unexpectedly high number of disagreements with the reference are masked, as
follows:
} The variant caller treats each insertion or deletion as a single mismatch.
} Base calls with more than 2 mismatches to the reference sequence within 20 bases of
the call are ignored.
} If the call occurs within the first or last 20 bases of a read, the mismatch limit is
applied to a 41-base window at the corresponding end of the read.
} The mismatch limit is applied to the entire read when the read length is 41 or
shorter.
Indel Genotypes
The variant caller filters out all bases marked by the mismatch density filter and any N
base calls that remain after the end-trimming step. These filtered base calls are not used
for site-genotyping but appear in the filtered base call counts in the variant caller output
for each site.
All remaining base calls are used for site-genotyping. The genotyping method
heuristically adjusts the joint error probability that is calculated from multiple
observations of the same allele on each strand of the genome. This correction accounts
for the possibility of error dependencies.
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Variant Call Output
After the SNV and indel genotyping methods are complete, the variant caller applies a
final set of heuristic filters to produce the final set of calls in the output.
The output in the genome variant call (gVCF) file captures the genotype at each position
and the probability that the consensus call differs from reference. This score is expressed
as a Phred-scaled quality score.
Variant Calling
Developed by Illumina, the somatic variant caller identifies variants present at low
frequency in the DNA sample.
The somatic variant caller identifies SNPs in 3 steps:
} Considers each position in the reference genome separately
} Counts bases at the given position for aligned reads that overlap the position
} Computes a variant score that measures the quality of the call using Poisson model.
Variants are first called for each pool separately. Then, variants from each pool are
compared and combined into a single output file. If a variant meets the following
criteria, the variant is marked as PASS in the variant call (VCF) file:
} The variant is present in both pools
} Has a cumulative depth of 1000 or an average depth of 500x per pool
} Has a variant frequency of ≥ 3% as reported in the merged VCF file
A locus for a mutation or reference is classified as a no call under the following
conditions:
} The variant frequency is near the signal noise level between 1% and 2.6%
} The variant quality is < Q30
} The depth is < 500
} Significant strand bias is detected
} The indel occurs in a homopolymer region
Illumina On-Node Annotation (IONA)
Annotation with IONA populates several values in the VCF file, including dbSNP ID (in
the ID column), and some values in the INFO column. More detailed and extensive
annotations are stored in a binary ANT file. This binary file can be imported into
VariantStudio. For more information, see
www.illumina.com/informatics/research/biological-datainterpretation/variantstudio.html.
Ensembl or RefSeq annotation through IONA is available for alignments against the
human reference genome: UCSC build hg19 / Ensembl build GRCh37 / NCBI build 37.2.
TruSeq Amplicon v2.0 BaseSpace App Guide
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Analysis Methods
This method treats the highest-quality base call from each allele and strand as an
independent observation and leaves the associated base call quality scores unmodified.
Quality scores for subsequent base calls for each allele and strand are then adjusted. This
adjustment is done to increase the joint error probability of the given allele above the
error expected from independent base call observations.
Analysis Output
To view the results, click the Projects tab, then the project name, and then the analysis.
Figure 2 Output Navigation Bar
After analysis is complete, access the output through the left navigation bar.
} Analysis Info—Information about the analysis session, including log files.
} Inputs—Lists the samples and settings specified for the analysis session.
} Output Files—Output files for the sample.
} Summary Analysis Report—Analysis metrics for the aggregate results, displayed
when multiple samples are analyzed.
} Sample Analysis Reports—Analysis reports for each sample.
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.
Session Type
Number of nodes used.
Status
Status of the analysis session. The status shows either Running
or Complete.
Log Files
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File Name
Description
AmpliconRunStatistics.xml
Provides amplicon-related information.
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Description
CompletedJobInfo.xml
Contains information about the completed analysis
session.
Logging.zip
Contains all detailed log files for each step of the
workflow.
RunInfo.xml
Identifies the boundaries of the reads (including index
reads).
SampleSheet.csv
Sample sheet.
SampleSheetUsed.csv
A copy of the sample sheet, generated at the beginning of
the workflow.
[Manifest name].txt
Manifest files used in the analysis.
Analysis Output
File Name
Output Files
The Output Files page provides access to the output files for each sample analysis.
} BAM Files—Aligned sequences and quality scores in the BAM (*.bam) file format.
} VCF Files—Variant calls in the VCF (*.vcf) file format.
} Genome VCF Files—Variants, references, and no calls for all sites in the genome in
the genome VCF (gVCF) file format.
} Annotation File—Detailed annotations in a binary file format.
} Summary File—Statistics for each sample.
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.
BAM files contain a header section and an alignments 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.
} 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.
TruSeq Amplicon v2.0 BaseSpace App Guide
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VCF File 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.
VCF files use the file naming format SampleName_S#.vcf, where # is the sample number
determined by the order that samples are listed for the run.
VCF File Header—Includes the VCF file format version and the variant caller version.
The header lists the annotations used in the remainder of the file. If MARS is listed, the
Illumina internal annotation algorithm annotated the VCF file. The VCF header includes
the reference genome file and BAM file. The last line in the header contains the column
headings for the data lines.
VCF File Data Lines—Each data line contains information about a single variant.
VCF File Headings
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Heading
Description
CHROM
The chromosome of the reference genome. Chromosomes appear in
the same order as the reference FASTA file.
POS
The single-base position of the variant in the reference chromosome.
For SNPs, this position is the reference base with the variant; for indels
or deletions, this position is the reference base immediately before the
variant.
ID
The rs number for the SNP obtained from dbSNP.txt, if applicable.
If there are multiple rs numbers at this location, the list is semicolon
delimited. If no dbSNP entry exists at this position, a missing value
marker ('.') is used.
REF
The reference genotype. For example, a deletion of a single T is
represented as reference TT and alternate T. An A to T single nucleotide
variant is represented as reference A and alternate T.
ALT
The alleles that differ from the reference read.
For example, an insertion of a single T is represented as reference A and
alternate AT. An A to T single nucleotide variant is represented as
reference A and alternate T.
QUAL
A Phred-scaled quality score assigned by the variant caller.
Higher scores indicate higher confidence in the variant and lower
probability of errors. For a quality score of Q, the estimated probability
of an error is 10-(Q/10). For example, the set of Q30 calls has a 0.1% error
rate. Many variant callers assign quality scores based on their statistical
models, which are high in relation to the error rate observed.
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Analysis Output
VCF File Annotations
Heading
Description
FILTER
If all filters are passed, PASS is written in the filter column.
• LowDP—Applied to sites with depth of coverage below a cutoff.
• LowGQ—The genotyping quality (GQ) is below a cutoff.
• LowQual—The variant quality (QUAL) is below a cutoff.
• LowVariantFreq—The variant frequency is less than the given
threshold.
• R8—For an indel, the number of adjacent repeats (1-base or 2-base)
in the reference is greater than 8.
• SB—The strand bias is more than the given threshold. Used with the
Somatic Variant Caller and GATK.
INFO
Possible entries in the INFO column include:
• AC—Allele count in genotypes for each ALT allele, in the same order
as listed.
• AF—Allele Frequency for each ALT allele, in the same order as listed.
• AN—The total number of alleles in called genotypes.
• CD—A flag indicating that the SNP occurs within the coding region
of at least 1 RefGene entry.
• DP—The depth (number of base calls aligned to a position and used
in variant calling).
• Exon—A comma-separated list of exon regions read from RefGene.
• FC—Functional Consequence.
• GI—A comma-separated list of gene IDs read from RefGene.
• QD—Variant Confidence/Quality by Depth.
• TI—A comma-separated list of transcript IDs read from RefGene.
FORMAT
The format column lists fields separated by colons. For example,
GT:GQ. The list of fields provided depends on the variant caller used.
Available fields include:
• AD—Entry of the form X,Y, where X is the number of reference calls,
and Y is the number of alternate calls.
• DP—Approximate read depth; reads with MQ=255 or with bad mates
are filtered.
• GQ—Genotype quality.
• GQX—Genotype quality. GQX is the minimum of the GQ value and
the QUAL column. In general, these values are similar; taking the
minimum makes GQX the more conservative measure of genotype
quality.
• GT—Genotype. 0 corresponds to the reference base, 1 corresponds
to the first entry in the ALT column, and so on. The forward slash (/)
indicates that no phasing information is available.
• NL—Noise level; an estimate of base calling noise at this position.
• PL—Normalized, Phred-scaled likelihoods for genotypes.
• SB—Strand bias at this position. Larger negative values indicate less
bias; values near 0 indicate more bias. Used with the Somatic Variant
Caller and GATK.
• VF—Variant frequency; the percentage of reads supporting the
alternate allele.
SAMPLE
The sample column gives the values specified in the FORMAT column.
TruSeq Amplicon v2.0 BaseSpace App Guide
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Genome VCF Files
Genome VCF (gVCF) files are VCF v4.1 files that follow a set of conventions for
representing all sites within the genome in a reasonably compact format. The gVCF files
include all sites within the region of interest in a single file for each sample.
The gVCF file shows no-calls at positions with low coverage, or where a low-frequency
variant (< 3%) occurs often enough (> 1%) that the position cannot be called to the
reference. A genotype (GT) tag of ./. indicates a no-call.
For more information, see sites.google.com/site/gvcftools/home/about-gvcf.
Annotation File
Illumina On-Node Annotation (IONA) generates a binary annotation file (*.ant), which
contains consequences for all affected transcripts. The annotations are more detailed than
the annotations in the VCF file. You can view this binary file in VariantStudio. For more
information, see www.illumina.com/informatics/research/biological-datainterpretation/variantstudio.html.
Summary File
The TruSeq Amplicon v2.0 App produces an overview of statistics for each sample and
the aggregate results in a comma-separated values (CSV) format: *.summary.csv. These
files are located in the results folder for each sample and the aggregate results.
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Statistic
Definition
Sample ID
IDs of samples reported in the file.
Sample Name
Names of samples reported in the file.
Run Folder
Run folders for samples reported in the file.
Reference genome
Reference genome selected.
Manifest
The manifest file used for analysis. This file specifies the
targeted regions for the aligner and variant caller.
Number of amplicon
regions
The number of amplicon regions that were sequenced.
Total length of
amplicon regions
The total length of the sequenced bases in the targeted region.
Total PF reads
The number of reads passing filter for the sample.
Total aligned reads
The total number of reads passing filter present in the data set
that aligned to the reference genome.
Numbers are calculated per read, and over both reads.
Percent aligned reads
The percentage of reads passing filter that aligned to the
reference genome.
Numbers are calculated per read, and over both reads.
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Definition
Total probe bases
Total number of bases that aligned to the probe sequences
(ULSO and DLSO) and are soft-clipped in the BAM files.
Numbers are calculated per read, and over both reads.
Total aligned nonprobe bases
Total number of bases that aligned to the reference, excluding
bases aligning to the probe sequences. This number is the
same as the number of bases aligned in the BAM file (probe
sequence bases are soft-clipped). Numbers are calculated per
read, and over both reads.
Total PF bases
The number of bases passing filter for the sample. Numbers
are calculated per read, and over both reads.
Percent Q30 bases
The percentage of bases with a quality score of 30 or higher.
Numbers are calculated per read, and over both reads.
Total aligned bases
The total number of bases present in the data set that aligned
to the reference genome.
Numbers are calculated per read, and over both reads.
Percent aligned bases
The percentage of bases that aligned to the reference genome.
Numbers are calculated per read, and over both reads.
Mismatch rate
The average percentage of mismatches across both reads 1
and 2 over all cycles.
Numbers are calculated per read.
Amplicon mean
coverage
The total number of aligned reads to the targeted region
divided by the number of targeted regions.
Uniformity of
Coverage (Pct >
0.2*mean)
The percentage of targeted base positions in which the read
depth is greater than 0.2 times the mean region target
coverage depth.
SNVs, Insertions,
Deletions (All)
Total number of variants present in the data set.
SNVs, Insertions,
Deletions
Total number of variants present in the data set that pass the
quality filters.
SNV Ts/Tv ratio
Transition rate of SNVs that pass the quality filters divided by
transversion rate of SNVs that pass the quality filters.
Transitions are interchanges of purines (A, G) or of
pyrimidines (C, T). Transversions are interchanges of purine
and pyrimidine bases (for example, A to T).
SNVs, Insertions,
Deletions Het/Hom
ratio
Number of heterozygous variants/Number of homozygous
variants.
SNVs, Insertions,
Deletions (Percent
found in dbSNP)
100*(Number of variants in dbSNP/Number of variants).
SNVs, Insertions,
Deletions in genes
Number of variants that fall into a gene.
TruSeq Amplicon v2.0 BaseSpace App Guide
Analysis Output
Statistic
15
Statistic
Definition
SNVs, Insertions,
Deletions in exons
Number of variants that fall into an exon.
SNVs, Insertions,
Deletions in coding
regions
Number of variants that fall into a coding region.
SNVs, Insertions,
Deletions in UTR
regions
Number of variants that fall into an untranslated region
(UTR).
SNVs, Insertions,
Deletions in splice site
regions
Number of variants that fall into a splice site region.
Stop gained SNVs,
Insertions, Deletions
Number of variants that cause an additional stop codon.
Stop lost SNVs,
Insertions, Deletions
Number of variants that cause the loss of a stop codon.
Non-synonymous
SNVs, Insertions,
Deletions
Number of variants that cause an amino acid change in a
coding region.
Synonymous SNVs
Number of variants that are within a coding region, but do
not cause an amino acid change.
Frameshift Insertions,
Deletions
Number of variants that cause a frameshift.
Sample Analysis Reports
The TruSeq Amplicon v2.0 App provides an overview of statistics per sample on the
Analysis Reports sample pages. To download statistics in the TruSeq Amplicon
Sequencing Report, click PDF Summary Report.
Amplicon Summary
Table 1 Amplicon Summary Table
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Statistic
Definition
Manifest (PDF only)
The name of the manifest used in the analysis.
Number of Amplicon
Regions
The number of amplicon regions that were sequenced.
Total Length of
Amplicon Regions
The total length of the sequenced bases in the targeted region.
Document # 15055857 v02
Analysis Output
Table 2 Read Level Statistics Table
Statistic
Definition
Total Aligned Reads
The total number of reads passing filter present in the data set
that aligned to the reference genome.
Percent Aligned Reads
The percentage of reads passing filter that aligned to the
reference genome.
Table 3 Base Level Statistics Table
Statistic
Definition
Percent Q30
The percentage of bases with a quality score of 30 or higher.
Total Aligned Bases
The total number of bases present in the data set that aligned
to the reference genome.
Percent Aligned Bases
The percentage of bases that aligned to the reference genome.
Mismatch Rate
The average percentage of mismatches across both reads 1
and 2 over all cycles.
Small Variants Summary
This table provides metrics about the number of SNVs, deletions, and insertions.
Table 4 Small Variants Summary Table
Statistic
Definition
Total Passing
The total number of variants present in the data set that
passed the variant quality filters.
Percent Found in
dbSNP
100*(Number of variants in dbSNP/Number of variants).
Het/Hom Ratio
Number of heterozygous variants/Number of homozygous
variants.
Ts/Tv Ratio
Transition rate of SNVs that pass the quality filters divided by
transversion rate of SNVs that pass the quality filters.
Transitions are interchanges of purines (A, G) or of
pyrimidines (C, T). Transversions are interchanges between
purine and pyrimidine bases (for example, A to T).
Table 5 Variants by Sequence Context Table
Statistic
Definition
Number in Genes
The number of variants that fall into a gene.
Number in Exons
The number of variants that fall into an exon.
Number in Coding
Regions
The number of variants that fall into a coding region.
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Statistic
Definition
Number in
UTR Regions
The number of variants that fall into an untranslated region
(UTR).
Number in Splice Site
Regions
The number of variants that fall into a splice site region.
To view the guidelines for calculating variation consequences, visit the Ensembl website:
uswest.ensembl.org/info/genome/variation/predicted_data.html#consequences.
Table 6 Variants by Consequence Table
Statistic
Definition
Frameshifts
The number of variants that cause a frameshift.
Non-synonymous
The number of variants that cause an amino acid change in a
coding region.
Synonymous
The number of variants that are within a coding region, but
do not cause an amino acid change.
Stop Gained
The number of variants that cause an additional stop codon.
Stop Lost
The number of variants that cause the loss of a stop codon.
Variants Table
Use the Variants Table to view, sort, filter, and export a subset of the data provided in
the VCF files.
NOTE
The Variants table is provided on the Sample Analysis Reports page, and not in the TruSeq
Amplicon Sequencing Report.
Table 7 Variants Table
18
Statistic
Definition
Chromosome (Chr)
Name of reference chromosome.
Position (Pos)
Position within reference chromosome.
Reference Allele (Ref)
The reference allele.
Variant Allele (Alt)
The alt allele.
Variant Type (Type)
Type of variant, including single nucleotide variant (SNV),
insertion, and deletion.
Sequence Context
(Context)
Location of the variant based on annotations of the reference
genome.
Consequence
Predicted transcript consequence as described at
uswest.ensembl.org/info/genome/variation/predicted_
data.html#consequences.
Document # 15055857 v02
Analysis Output
Statistic
Definition
dbSNP ID (dbSNP)
Identifier in the Single Nucleotide Polymorphism Database
(dbSNP), a free public archive for genetic variation within and
across different species developed and hosted by the National
Center for Biotechnology Information (NCBI).
COSMIC ID
(COSMIC)
The numeric identifier for the variant in the Catalogue of
Somatic Mutations in Cancer (COSMIC) database.
ClinVar
Clinical significance based on the freely accessible, public
archive of reports of the relationships among human
variations and phenotypes
Variant Quality (Qual)
Phred-scaled quality score indicating how confident we are in
this asserted haplotype.
Variant Frequency (Alt
Freq)
Proportion of the variant allele among all alleles being
considered.
Total Depth
Number of reads aligned at this position.
Reference Allele
Depth (Ref Depth)
Number of reads containing the reference allele.
Variant Allele Depth
(Alt Depth)
Number of reads containing the variant allele.
Strand Bias
Strand bias is a type of sequencing bias in which 1 DNA strand
is favored over the other, which can result in incorrect
evaluation of the amount of evidence observed for one allele
versus the other.
Coverage Summary
Table 8 Coverage Summary Table
Statistic
Definition
Amplicon Mean
Coverage
The total number of aligned reads to the targeted region
divided by the number of targeted regions.
Uniformity of
Coverage (Pct
> 0.2*mean)
The percentage of targeted base positions in which the read
depth is greater than 0.2 times the mean region target
coverage depth.
Coverage by Amplicon Region Plot
The Coverage by Amplicon Region plot shows the number of bases plotted against the
amplicon region. It has the following features:
} Amplicon regions with coverage values less than the low coverage threshold (0.2 *
amplicon mean coverage) are highlighted in red.
} The horizontal red line marks the low coverage threshold.
} The orange line marks the moving average of all coverage values.
} Off-targets are excluded in the plot.
TruSeq Amplicon v2.0 BaseSpace App Guide
19
Figure 3 Example Coverage by Amplicon Region Plot
Coverage values for each amplicon are detailed in the downloadable Export (CSV) file.
Summary Analysis Report
The TruSeq Amplicon v2.0 App provides an Aggregate Summary for all samples.
Statistics are plotted against samples with tables providing additional metrics.
Amplicon Summary
Table 9 Read Level Statistics Table
Statistic
20
Definition
Total Aligned Reads
(R1/R2)
The total number of reads passing filter present in the data set
that aligned to the reference genome.
Numbers are per read.
Percent Aligned Reads
(R1/R2)
The percentage of reads passing filter that aligned to the
reference genome.
Numbers are calculated per read.
Overall Aligned Reads
The percentage of reads passing filter that aligned for the
sample across both reads.
Document # 15055857 v02
Analysis Output
Table 10 Base Level Statistics Table
Statistic
Definition
Total Aligned Bases
(R1/R2)
The total number of bases present in the data set that aligned
to the reference genome.
Numbers are calculated per read.
Overall Total Aligned
Bases
The total number of bases present in the data set that aligned
to the reference genome across both reads (R1 and R2). The
value is the sum of the individual Total Aligned Bases values.
Percent Aligned Bases
(R1/R2)
The percentage of bases that aligned to the reference genome.
Numbers are calculated per read, and the average over both
reads.
Overall Percent
Aligned Bases
The percentage of bases that aligned to the reference genome
across both reads (R1 and R2). The value is the average of the
individual Percent Aligned Bases values.
Percent Q30 (R1/R2)
The percentage of bases with a quality score of 30 or higher.
Numbers are calculated per read.
Mismatch Rate
(R1/R2)
The average percentage of mismatches across both reads 1
and 2 over all cycles.
Numbers are calculated per read.
Small Variants Summary
Table 11 SNVs Table
Statistic
Definition
SNVs
The total number of SNVs present in the data set that passed
the variant quality filters.
SNV Ts/Tv Ratio
Transition rate of SNVs that pass the quality filters divided by
transversion rate of SNVs that pass the quality filters.
Transitions are interchanges of purines (A, G) or of
pyrimidines (C, T). Transversions are interchanges between
purine and pyrimidine bases (for example, A to T).
SNV Het/Hom Ratio
Number of heterozygous variants/Number of homozygous
variants.
Table 12 Insertions Table
Statistic
Definition
Insertions
The total number of insertions present in the data set that
passed the variant quality filters.
Insertion Het/Hom
Ratio
Number of heterozygous variants/Number of homozygous
variants.
TruSeq Amplicon v2.0 BaseSpace App Guide
21
Table 13 Deletions Table
Statistic
Definition
Deletions
The total number of deletions present in the data set that
passed the variant quality filters.
Deletions Het/Hom
Ratio
Number of heterozygous variants/Number of homozygous
variants.
Coverage Summary
Table 14 Coverage Summary Table
22
Statistic
Definition
Amplicon Mean
Coverage Depth
The total number of aligned reads to the targeted region
divided by the number of targeted regions.
Document # 15055857 v02
Revision History
Revision History
Date
Document
Description of Change
Document #
15055857 v02
January
2016
Reorganized topics, updated writing style.
Document #
15055857 v01
October
2015
Release supporting TruSeq Amplicon v2.0.
TruSeq Amplicon v2.0 BaseSpace App Guide
23
Notes
For technical assistance, contact Illumina Technical Support.
Table 15 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 16 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Japan
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
China
400.635.9898
Singapore
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
Taiwan
Hong Kong
800960230
United Kingdom
Ireland
1.800.812949
Other countries
Italy
800.874909
Contact Number
0800.111.5011
0800.0223859
0800.451.650
800.16836
1.800.579.2745
900.812168
020790181
0800.563118
00806651752
0800.917.0041
+44.1799.534000
Safety data sheets (SDSs)—Available on the Illumina website at
support.illumina.com/sds.html.
Product documentation—Available for download in PDF from the Illumina website. Go
to support.illumina.com, select a product, then select Documentation & Literature.
TruSeq Amplicon v2.0 BaseSpace App Guide
Technical Assistance
Technical Assistance
Illumina
5200 Illumina Way
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com
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