Output Files

The following section describes the outputs produced by DRAGEN Array.

CNV VCF File

DRAGEN Array produces one CNV variant call file (VCF) (*.cnv.vcf) per sample to report the CN status on the gene and sub gene level, along with the CN events for PGx targets.

The CNV VCF output file follows the standard VCF format. The QUAL field in the VCF file measures the CNV call quality. The CNV call quality is a Phred-scaled score capped at 60 and the minimal value is 0. Low quality calls (QUAL<7) are flagged by the Q7 filter. Low quality samples with LogRDev greater than a threshold 0.2 are flagged with the SampleQuality flag.

The CNV VCF files are by default bgzipped (Block GZIP) and have the “.gz” extension. The compression saves storage space and facilitates efficient lookup when indexed with the TBI Index File. To view these files as plain text, they can be uncompressed with bgzip from Samtools or other third-party tools. The CNV VCF must be bgzipped and indexed to be used in downstream DRAGEN Array commands, such as star allele calling.

The CNV VCF output file includes the following content.

##fileformat=VCFv4.1

##source=dragena 1.0.0

##genomeBuild=38

##reference=file:///hg38_with_alt/hg38_nochr_MT.fa

##FORMAT=<ID=CN,Number=1,Type=Integer,Description="Copy number genotype for imprecise events. CN=5 indicates 5 or 5+">

##FORMAT=<ID=NR,Number=1,Type=Float,Description="Aggregated normalized intensity">

##ALT=<ID=CNV,Description="Copy number variant region">

##FILTER=<ID=Q7,Description="Quality below 7">

##FILTER=<ID=SampleQuality,Description="Sample was flagged as potentially low-quality due to high noise levels.">

##INFO=<ID=CNVLEN,Number=1,Type=Integer,Description="Number of bases in CNV hotspot">

##INFO=<ID=PROBE,Number=1,Type=Integer,Description="Number of probes assayed for CNV hotspot">

##INFO=<ID=END,Number=1,Type=Integer,Description="End position of CNV hotspot">

##INFO=<ID=SVTYPE,Number=1,Type=String,Description="Structural Variant Type">

##CNVOverallPloidy=1.8

##CNVGCCorrect=True

##contig=<ID=1,length=248956422>

##contig=<ID=4,length=190214555>

##contig=<ID=10,length=133797422>

##contig=<ID=16,length=90338345>

##contig=<ID=19,length=58617616>

##contig=<ID=22,length=50818468>

##contig=<ID=22_KI270879v1_alt,length=304135>

#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT 204619760001_R01C01

1 109687842 CNV:GSTM1:chr1:109687842:109693526 N <CNV> 60 PASS CNVLEN=5685;PROBE=124;END=109693526;SVTYPE=CNV CN:NR 2:0.966631132771593

4 68537222 CNV:UGT2B17:chr4:68537222:68568499 N <CNV> 60 PASS CNVLEN=31278;PROBE=383;END=68568499;SVTYPE=CNV CN:NR 0:0.376696837881692

10 133527374 CNV:CYP2E1:chr10:133527374:133539096 N <CNV> 60 PASS CNVLEN=11723;PROBE=194;END=133539096;SVTYPE=CNV CN:NR 2:0.980059731860893

16 28615068 CNV:SULT1A1:chr16:28615068:28623382 N <CNV> 57 PASS CNVLEN=8315;PROBE=164;END=28623382;SVTYPE=CNV CN:NR 2:0.980552325552963

19 40844791 CNV:CYP2A6.intron.7:chr19:40844791:40845293 N <CNV> 60 PASS CNVLEN=503;PROBE=38;END=40845293;SVTYPE=CNV CN:NR 2:0.9663775484762

19 40850267 CNV:CYP2A6.exon.1:chr19:40850267:40850414 N <CNV> 60 PASS CNVLEN=148;PROBE=21;END=40850414;SVTYPE=CNV CN:NR 2:0.9663775484762

22 42126498 CNV:CYP2D6.exon.9:chr22:42126498:42126752 N <CNV> 48 PASS CNVLEN=255;PROBE=370;END=42126752;SVTYPE=CNV CN:NR 2:0.981703411438716

22 42129188 CNV:CYP2D6.intron.2:chr22:42129188:42129734 N <CNV> 10 PASS CNVLEN=547;PROBE=333;END=42129734;SVTYPE=CNV CN:NR 2:0.965498002434641

22 42130886 CNV:CYP2D6.p5:chr22:42130886:42131379 N <CNV> 60 PASS CNVLEN=494;PROBE=172;END=42131379;SVTYPE=CNV CN:NR 2:0.970341562236357

22_KI270879v1_alt 270316 CNV:GSTT1:chr22_KI270879v1_alt:270316:278477 N <CNV> 60 PASS CNVLEN=8162;PROBE=91;END=278477;SVTYPE=CNV CN:NR 2:1.01191145130511

SNV VCF File

The software produces one genotyping variant call file (*.snv.vcf) file per sample, covering single nucleotide variants (SNV) and indels for the sample. It reports GenCell score (GS), B Allele Frequency (BAF), and Log R Ratio (LRR) per variant.

Certain SNV and indel calls can be skipped when reported in the VCF. Skipped data can include unmapped loci, intensity-only probes used for CNV identification, and indels that do not map back to the genome. See Warning/Error Messages and Logs for messages that may be seen with DRAGEN Array Local related to the skipped data.

The BAF and LRR are oriented with Ref as A and Alt as B relative to the reference genome, while GS is agnostic to the reference genome. Users familiar with GenomeStudio may observe BAF and LRR reported in the VCF as 1 minus the value reported in GenomeStudio depending on the Ref Alt allele orientation with the reference genome. GenomeStudio reports these values based on the information in the manifest without knowledge of the reference genome.

The SNV VCF files are by default bgzipped (Block GZIP) and have the “.gz” extension. The compression saves storage space and facilitates efficient lookup when indexed with the TBI Index File. To view these files as plain text, they can be uncompressed with bgzip from Samtools or other third-party tools. The SNV VCF must be bgzipped and indexed to be used in downstream DRAGEN Array commands, such as star allele calling.

The SNV VCF output file includes the following content. The last row shows an example of variant call.

##fileformat=VCFv4.1

##source=dragena 1.0.0

##genomeBuild=38

##reference=file:///genomes/38/genome.fa

##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">

##FORMAT=<ID=GS,Number=1,Type=Float,Description="GenCall score. For merged multi-assay or multi-allelic records, min GenCall score is reported.">

##FORMAT=<ID=BAF,Number=1,Type=Float,Description="B Allele Frequency">

##FORMAT=<ID=LRR,Number=1,Type=Float,Description="LogR ratio">

##contig=<ID=1,length=248956422>

##contig=<ID=2,length=242193529>

##contig=<ID=3,length=198295559>

##contig=<ID=4,length=190214555>

##contig=<ID=5,length=181538259>

##contig=<ID=6,length=170805979>

##contig=<ID=7,length=159345973>

##contig=<ID=8,length=145138636>

##contig=<ID=9,length=138394717>

##contig=<ID=10,length=133797422>

##contig=<ID=11,length=135086622>

##contig=<ID=12,length=133275309>

##contig=<ID=13,length=114364328>

##contig=<ID=14,length=107043718>

##contig=<ID=15,length=101991189>

##contig=<ID=16,length=90338345>

##contig=<ID=17,length=83257441>

##contig=<ID=18,length=80373285>

##contig=<ID=19,length=58617616>

##contig=<ID=20,length=64444167>

##contig=<ID=21,length=46709983>

##contig=<ID=22,length=50818468>

##contig=<ID=MT,length=16569>

##contig=<ID=X,length=156040895>

##contig=<ID=Y,length=57227415>

#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT 202937470021_R06C01

1 2290399 rs878093 G A . PASS . GT:GS:BAF:LRR 0/1:0.7923:0.50724137:0.14730307

Genotype Call (GTC) File

The genotype call algorithm produces one genotype call file (.gtc) per sample analyzed. The Genotype Call (GTC) file contains the small variant (SNV and indel) genotype for each marker specified by the product and sample quality metrics. The sample marker location is not included and must be extracted from the manifest file. Binary proprietary format can be parsed using the Illumina open-source tool BeadArray Library File Parser.

BedGraph File

The BedGraph file contains the log R ratios from the genotyping algorithm for use in visual tools.

Star Allele CSV File

The Star Allele CSV file is an intermediate file generated by the star-allele call command and serves as the input to the star-allele annotate command. It contains all the star allele calls for all samples in a run. Each row in the file provides either a star allele diplotype or simple variant call for a PGx-related gene. Star allele diplotype calls for a sample and a gene may span multiple lines where alternative solutions can be listed.

The Star Allele CSV file also contains meta information marked by # at the top of the file for the genome build and PGx database used for the star allele calling.

The star_allele.csv file contains the following details per sample:

Field	Description
Sample	Sentrix barcode and position of the sample.
Rank	Rank of a single star allele solution for a gene. The top solution based on quality score is ranked as 1 with the alternative solutions ranked lower.
Gene or Variant	The gene symbol, or gene symbol plus rsID for variants.
Type	‘Haplotype’ (star allele) or ‘Variant’ PGx calling type.
Solution	Star allele or variant solution. If diploid, variant solutions have the format of Allele1/Allele2.
Solution Long	Long format solution for star alleles. The field has the following format: Structural Variant Type: Underlying Star allele. An example of a long solution is: Complete: CYP2D64, Complete: CYP2D610, CYP2D668: CYP2D64 where there are two complete alleles that have CYP2D64 and CYP2D610 haplotypes and one CYP2D668 structural variant that has a CYP2D64 haplotype configuration.
Supporting Variants	All variants present in the array that support the star allele solution. The field has the following format: Long Solution Star Allele: (Supporting Variants). Each supporting variant is listed with essential information extracted from the SNV VCF to assist with troubleshooting, including Chromosome, Location, Reference allele, Alternative allele, Genotype, GenCall score (GS), and B-allele frequency (BAF).
Missing/Masked Core Variants	All variants not present in the array or not called in the SNV VCF file for the star allele. The field has the following format: Long Solution Star-Allele: (Missing Variants).
All Missing Variants in Array	All core definition variants that are not on the array or are not called in the SNV VCF along with the associated star alleles that are impacted. The field has the following format: Missing Variant: (List of impacted star alleles).
Collapsed Star-Alleles	Star alleles that cannot be distinguished from the solution star allele given the input array’s content. The field has the following format: Long Solution Star-Allele: (List of collapsed star alleles). The most frequent star allele based on the population frequency of PGx alleles will be the star allele in the solution.
Score	Quality score of the solution including the population frequency of PGx alleles. The score ranges from 0 to 1.
Raw Score:	Raw quality score of the solution without including the population frequency of PGx alleles. The score ranges from 0 to 1.
Copy Number Solution	Estimated copy number for each gene region. The field has the following format: Gene Region: Copy Number.

Below is an example of the first 4 columns from a star allele CSV file:

Sample,Rank,Gene or Variant,Type,Solution

204650490282_R02C01,1,CYP2C9,Haplotype,*9/*11

204650490282_R02C01,1,CYP2C19,Haplotype,*2/*10

Genotype Summary Files

The software produces genotype summary files (gt_sample_summary.csv and gt_sample_summary.json) that contains the following details per sample:

• Sample ID

• Sample Name

• Sample Folder

• Autosomal Call Rate

• Call Rate

• Log R Ratio Std Dev

• Sex Estimate

• TGA_Ctrl_5716 Norm R

The TGA_Ctrl_5716 Norm R field is specific to PGx products (e.g., Global Diversity Array with enhanced PGx). The field value is the Normalized R value of one probe and is meant as an assay control where < 1 indicates the sample failed in the TGA (Targeted Gene Amplification) process. If the product does not have this probe, it is not included in the gt_sample_summary.

Final Report

DRAGEN Array Cloud produces a Final Report (gtc_final_report.csv) per analysis batch similar to the one available in GenomeStudio. It contains the following details per locus per sample:

Field	Description
SNP Name	SNP identifier.
SNP	SNP alleles as reported by assay probes. Alleles on the Design strand (the ILMN strand) are listed in order of Allele A/B.
Sample ID	Sample identifier.
Allele 1 – Top	Allele 1 corresponds to Allele A and are reported on the Top strand.
Allele 2 – Top	Allele 2 corresponds to Allele B and are reported on the Top strand.
Allele 1 – Forward	Allele 1 corresponds to Allele A and are reported on the Forward strand.
Allele 2 – Forward	Allele 2 corresponds to Allele B and are reported on the Forward strand.
Allele 1 – Plus	Allele 1 corresponds to Allele A and are reported on the Plus strand.
Allele 2 – Plus	Allele 2 corresponds to Allele B and are reported on the Plus strand.
GC Score	Quality metric calculated for each genotype (data point), and ranges from 0 to 1.
GT Score	The SNP cluster quality. Score for a SNP from the GenTrain clustering algorithm.
Log R Ratio	Base-2 log of the normalized R value over the expected R value for the theta value (interpolated from the R-values of the clusters). For loci categorized as intensity only; the value is adjusted so that the expected R value is the mean of the cluster.
B Allele Freq	B allele frequency for this sample as interpolated from known B allele frequencies of 3 canonical clusters: 0, 0.5 and 1 if it is equal to or greater than the theta mean of the BB cluster. B Allele Freq is between 0 and 1, or set to NaN for loci categorized as intensity only.
Chr	Chromosome containing the SNP.
Position	SNP chromosomal position.

Note: Analyses on products with large numbers of loci (>1 Million) and large numbers of samples (>100) yield a large (50+ Gigabyte) Final Report that are difficult to download and review. It’s recommended to create analysis configurations that do not produce this report if large batches are desired.

For more information on interpreting DNA strand and allele information, see Illumina Knowledge article How to interpret DNA strand and allele information for Infinium genotyping array data.

Locus Summary

DRAGEN Array Cloud produces a Locus Summary (locus_summary.csv) per analysis batch similar to the one available in GenomeStudio. It contains the following details per locus:

Field	Description
Locus_Name	Locus name from the manifest file.
Illumicode_Name	Locus ID from the manifest file.
#No_Calls	Number of loci with GenCall scores below the call region threshold.
#Calls	Number of loci with GenCall scores above the call region threshold.
Call_Freq	Call frequency or call rate calculated as follows: #Calls/(#No_Calls + #Calls)
A/A_Freq	Frequency of homozygote allele A calls.
A/B_Freq	Frequency of heterozygote calls.
B/B_Freq	Frequency of homozygote allele B calls.
Minor_Freq	Frequency of the minor allele.
Gentrain_Score	Quality score for samples clustered for this locus.
50%_GC_Score	50th percentile GenCall score for all samples.
10%_GC_Score	10th percentile GenCall score for all samples.
Het_Excess_Freq	Heterozygote excess frequency, calculated as (Observed -Expected)/Expected for the heterozygote class. If $f_{ab}$ is the heterozygote frequency observed at a locus, and p and q are the major and minor allele frequencies, then het excess calculation is the following: $(f_{ab} - 2pq)/(2pq + \varepsilon)$
ChiTest_P100	Hardy-Weinberg p-value estimate calculated using genotype frequency. The value is calculated with 1 degree of freedom and is normalized to 100 individuals.
Cluster_Sep	Cluster separation score.
AA_T_Mean	Normalized theta angles mean for the AA genotype.
AA_T_Std	Normalized theta angles standard deviation for the AA genotype.
AB_T_Mean	Normalized theta angles mean for the AB genotype.
AB_T_Std	Standard deviation of the normalized theta angles for the AB genotype.
BB_T_Mean	Normalized theta angles mean for the BB genotypes.
BB_T_Std	Standard deviation of the normalized theta angles for the BB genotypes.
AA_R_Mean	Normalized R value mean for the AA genotypes.
AA_R_Std	Standard deviation of the normalized R value for the AA genotypes.
AB_R_Mean	Normalized R value mean for the AB genotypes.
AB_R_Std	Standard deviation of the normalized R value for the AB genotypes.
BB_R_Mean	Normalized R value mean for the BB genotypes.
BB_R_Std	Standard deviation of the normalized R value for the BB genotypes.
Plus/Minus Strand	Designated "+" or "-" with respect to the reference genome strand. "U" designates unknown.

CN Summary File

The sample summary contains per sample key stats for each sample in a batch that contains the following details per sample:

• Sample ID

• Sample Name

• Sample Folder

Copy Number Batch File

The copy number batch summary file (cn_batch_summary.csv) shows the total copy number gain, loss, and neutral (CN=2) values for each target region across all the samples in the analysis.

Example copy number batch summary file content:

Target Region,Total CN gain,Total CN loss,Total CN neutral

CYP2A6.exon.1,0,1,47

CYP2A6.intron.7,0,1,47

CYP2D6.exon.9,2,4,42

CYP2D6.intron.2,7,2,39

CYP2D6.p5,13,2,33

CYP2E1,2,0,46

GSTM1,0,42,6

GSTT1,0,33,15

SULT1A1,0,0,48

UGT2B17,0,34,14

All Target Regions,24,119,337

Warning/Error Messages and Logs

The following scenarios result in a warning or error message:

• Manifest file used to generate GTC is not the same as the manifest file used to generate the CN model.

• FASTA files and FASTA index files do not match.

For the following scenarios, the software reports messages to the terminal output (as either a warning or an error):

• Indel processing for GTC to VCF conversion failed.

• The input folder does not contain the required input files.

• An input file is corrupt.

Examples of such notifications can include the following:

Error	Type	Cause
Reference allele is not queried for locus: {identifier}	Warning	True reference allele does not match any alleles in the manifest. The error is common for MNVs and will be addressed in future versions of the software.
Skipping non-mapped locus: {identifier}	Warning	Locus has no chromosome position (usually 0) These loci may be used for quality purposes or CNV calling only.
Skipping intensity only locus: {identifier}	Warning	Similar to non-mapped loci, intensity only probes have applications outside creating variants for SNV VCFs such as CNV calling.
Skipping indel: {identifier}	Warning	Indel context (deletion/insertion) could not be determined.
Failed to process entry for record: {identifier}	Warning	Unable to determine reference allele for indel.
Incomplete match of source sequence to genome for indel: {identifier}	Warning	Indel not properly mapped to the reference genome.
Failed to combine genotypes due to ambiguity - exm1068284 (InfiniumII): TT, ilmnseq_rs1131690890_mnv (InfiniumII): AA, rs1131690890_mnv (InfiniumII): AA	Warning	Detailed information about a NoCall ("./.”) in the VCF as a result of combining multiple probes that assay the same variant with conflicting results. The example here is two probes with homozygous REF genotypes (AA) and one probe with homozygous ALT probe (TT)
{numPassingSamples} sample(s) passed QC. " + Requires at least {minPassingSamples} samples to proceed.	Error	CNV calling is batch dependent and requires a certain number of samples with high-quality to make accurate calls. More high-quality samples need to be added to analysis batch to resolve error.
Invalid manifest file path {manifestPath}	Error	Application could not find manifest file provided or user error.
Failed to load cluster file: {e.Message}	Error	Corrupt or unreachable cluster file.

Star allele JSON File

The star allele JSON file is produced per sample. It contains the fields present in the star allele CSV file as well as additional meta data and annotations.

Fields included in the star allele JSON header are described below.

Field	Description
softwareVersion	DRAGEN Array software version, e.g. dragena 1.0.0.
genomeBuild	Genome build, e.g hg38.
databaseSources	Public databases with versions used as the sources of the star allele definitions, phenotypes, and population frequencies.
mappingFile	The PGx database file used for the star allele calling.
pgxGuideline	The PGx guidelines used for metabolizer status/phenotype annotations, e.g. CPIC or DPWG
sampleId	Sentrix barcode and position of the sample.
locusAnnotations	The star allele call information.

Fields included in the star allele call (locusAnnotations) information are described below.

Field	Description
gene	The gene symbol.
callType	‘Star Allele’ or ‘Variant’ PGx calling type.
genotype	Most likely star allele or variant solution. If diploid, variant solutions have the format of Allele1/Allele2.
activityScore	Activity score annotation of the determined genotype of the gene determined based on public PGx guidelines CPIC or DPWG.
phenotype	Metabolizer status and function annotations of the determined genotype of the gene based on lookup into public PGx guidelines CPIC or DPWG per user choice.
qualityScore	Quality score of the solution including the population frequency of PGx alleles. The score ranges from 0 to 1.
rawScore	Raw quality score of the solution without including the population frequency of PGx alleles. The score ranges from 0 to 1.
supportingVariants	All variants present in the array that support the star allele solution. The field the following format: Long Solution Star Allele: (Supporting Variants). Each supporting variant is listed with essential information extracted from the SNV VCF to assist with troubleshooting, including Chromosome, Location, Reference allele, Alternative allele, Genotype, GenCall score (GS), and B-allele frequency (BAF).
candidateSolutions	The set of alternative star allele calling solutions, this is only relevant for genes of the ‘Star Allele’ call type.
allMissingVariants	All core variants that are not available (e.g. not on the array, or no calls in the SNV VCF) for star allele calling for this gene. For star alleles, the field has the following format: Missing Variant: (List of impacted star alleles).
allelesTested	Alleles that are covered by the star allele caller. The capability to call star alleles is also dependent on array content coverage and data quality. This field is defined by the array's content and will be the same across all samples.
Fields included in the candidateSolution section, only available for star allele call type, are described below.

Field	Description
rank	Rank of a single star allele solution for a gene. The top solution based on quality score is ranked as 1 with the alternative solutions ranked lower.
genotype	Star allele or variant solution. If diploid, variant solutions have the format of Allele1/Allele2.
activityScore	Activity score annotation of the determined genotype of the gene determined based on public PGx guidelines CPIC or DPWG.
phenotype	Metabolizer status and function annotations of the determined genotype of the gene based on lookup into public PGx guidelines CPIC or DPWG per user choice.
qualityScore	Quality score of the solution including the population frequency of PGx alleles. The score ranges from 0 to 1.
rawScore	Raw quality score of the solution without including the population frequency of PGx alleles. The score ranges from 0 to 1.
alleles	The composite alleles of the candidate genotype solution.
solutionLong	Long format solution for star alleles. The field has the following format: Structural Variant Type: Underlying Star allele. An example of a long solution is: Complete: CYP2D64, Complete: CYP2D610, CYP2D668: CYP2D64 where there are two complete alleles that have CYP2D64 and CYP2D610 haplotypes and one CYP2D668 structural variant that has a CYP2D64 haplotype configuration.
supportingVariants	All variants present in the array that support the star allele solution. The field the following format: Long Solution Star Allele: (Supporting Variants). Each supporting variant is listed with essential information extracted from the SNV VCF to assist with troubleshooting, including Chromosome, Location, Reference allele, Alternative allele, Genotype, GenCall score (GS), and B-allele frequency (BAF).
missingVariants	All variants not present in the array or not called in the SNV VCF file for the star allele solution. The field has the following format: Long Solution Star-Allele: (Missing Variants).
collapsedAlleles	Star alleles that cannot be distinguished from the solution star allele given the input array’s content. The field has the following format: Long Solution Star-Allele: (List of collapsed star alleles). The most frequent star allele based on the population frequency of PGx alleles will be the star allele in the solution.
copyNumberRegions	Gene regions for the copy numbers listed in CopyNumberSolution.
copyNumberSolution	Estimated copy number for each gene region listed in CopyNumberRegions

Example of JSON file content:

{

"softwareVersion": "dragena 1.0.0",

"genomeBuild": "hg38",

"databaseSources": "PharmVar Version: 6.0.5, PharmGKB Database Version: Snapshot-2023.08.30, CPIC Database Version: 1.30.0",

"mappingFile": "gda_mapping_53e0931.zip",

"pgxGuideline": "CPIC",

"sampleId": "204619760027_R01C01",

"locusAnnotations": [

{

"gene": "CYP2C9",

"callType": "Star Allele",

"genotype": "*1/*1",

"activityScore": "2",

"phenotype": "Normal Metabolizer",

"qualityScore": "0.9999",

"rawScore": "0.9999",

"supportingVariants": "Complete: *1 ( )",

"candidateSolutions": [

{

"rank": 1,

"genotype": "*1/*1",

"activityScore": "2",

"phenotype": "Normal Metabolizer",

"qualityScore": 0.9999,

"rawScore": 0.9999,

"alleles": [

{

"solutionLong": "Complete: *1",

"supportingVariants": "Complete: *1 ( )",

"missingVariants": "Complete: *1 ( )",

"collapsedAlleles": "Complete: *1 ( )"

}

],

"copyNumberRegions": "p5,exon.1,intron.1,exon.2,intron.2,exon.3,intron.3,exon.4,intron.4,exon.5,intron.5,exon.6,intron.6,exon.7,intron.7,exon.8,intron.8,exon.9,p3",

"copyNumberSolution": "2,2,2,2,2,2,2,2,2,2,2,2,2,2,2,2,2,2,2"

}

],

TBI Index File

The TBI (TABIX) index file is associated with the bgzipped VCF files. It allows for data line lookup in VCF files for quick data retrieval. The format is a tab-delimited genome index file developed by Samtools as part of the HTSlib utilities. For more information, visit the Samtools website.

Methylation Control Probe Output File

The software produces a control probe output file ({BeadChipBarcode}_{Position}_ctrl.tsv.gz) per sample that includes the raw methylated and unmethylated values for each control probe.

Each control probe has an address, type, color channel, name, and probe ID. It also provides the raw signal for methylated green (MG), methylated red (MR), unmethylated green (UG) and unmethylated red (UR).

The file can help identify which probes are available on a given BeadChip.

Methylation CG Output File

The software produces a CG output file ({BeadChipBarcode}_{Position}_cgs.tsv.gz) per sample that includes beta values, m-values and detection p-values for each CG site.

Beta values measure methylation levels in a linear fashion for easy interpretation. Unmethylated probes are close to zero and methylated probes are close to 1.

M-values are a log transformed beta value which provides a more representative measure of methylation.

Detection p-values measure the likelihood that the signal is background noise. It is recommended that p-value >0.05 are excluded from analysis as they are likely background noise.

Methylation Sample QC Summary Files

The software produces methylation sample QC summary in .xlsx and .tsv file formats (sample_qc_summary.xlsx and sample_qc_summary.tsv) per analysis batch, which provides per sample QC data for all samples in the batch.

The QC summary provides details on 21 controls metrics (see tables below), which are computed in same way as in the BeadArray Control Reporter software from Illumina. In addition, it provides average red and green raw and normalized signals, time of scanning, proportion of probes passing, overall sample pass/fail status, and the failure codes for control metrics that did not pass. The sample pass status is defined as the passing of all 21 control metrics. The QC summary .xlsx file further highlights failing parameters for easy viewing.

The QC summary files contain the following fields:

Field	Description
Sentrix_ID	12-digit BeadChip Barcode associated with the sample.
Sentrix_Position	Row and column on the BeadChip ie R01C01
Sample_ID	Optional field that can be indicated using IDAT Sample Sheet
User Defined Meta Data	Optional field(s) that can be indicated using IDAT Sample Sheet. Any number of fields indicated will appear in this output file.
restoration	The default threshold is 0. If using the FFPE DNA Restore Kit, the restoration control identifies success of the FFPE restoration chemistry. Change the threshold from 0 to 1 if the FFPE DNA Restore Kit was used. The green channel intensity is higher than Background. Therefore, the metric provided is the Green Channel Intensity/Background.
staining_green staining_red	Staining controls are used to examine the efficiency of the staining step in both the red and green channels. These controls are independent of the hybridization and extension step. The green channel shows a higher signal for biotin staining when compared to biotin background, whereas the red channel shows higher signal for DNP staining when compared to DNP background. The metric provided for green is the (Biotin High value)/ (Biotin Bkg) and the metric provided for red is (DNP High value)/(DNP Bkg value) The default threshold is 5. This threshold can be increased on some scanners.
extension_green extension_red	Extension controls test the extension efficiency of A, T, C, and G nucleotides from a hairpin probe, and are therefore sample independent. In the green channel, the lowest intensity for C or G is always greater than the highest intensity for A or T. The metric provided is the (lowest of the C or G intensity)/ (highest of A or T extension) for a single sample. The default threshold is 5. This threshold can be increased on some scanners.
hybridization_high_medium hybridization_medium_low	Hybridization controls test the overall performance of the Infinium Assay using synthetic targets instead of amplified DNA. These synthetic targets complement the sequence on the array, allowing the probe to extend on the synthetic target as a template. Synthetic targets are present in the Hybridization Buffer at 3 levels, monitoring the response from high-concentration (5 pM), medium concentration (1 pM), and low concentration (0.2 pM) targets. All bead type IDs result in signals with various intensities, corresponding to the concentrations of the initial synthetic targets. The value for high concentration is always higher than medium and the value for medium concentration is always higher than low. The metric provided is the value of high/medium and the value of medium/low. The default thresholds are 1. Do not change the default threshold.
target_removal1 target_removal2	Target removal controls test the efficiency of the stripping step after the extension reaction. In contrast to allele-specific extension, the control oligos are extended using the probe sequence as a template. This process generates labeled targets. The probe sequences are designed such that extension from the probe does not occur. All target removal controls result in low signal compared to the hybridization controls, indicating that the targets were removed efficiently after extension. Target removal controls are present in the Hybridization Buffer. The Background for the same sample is close to or larger than either control. The metric provided is Background/Control Intensity. The default threshold is 1. Do not change the default threshold; however, the offset correction can be changed.
bisulfite_conversion1_green bisulfite_conversion1_background_green bisulfite_conversion1_red bisulfite_conversion1_background_red	These controls assess the efficiency of bisulfite conversion of the genomic DNA. The Infinium Methylation probes query a [C/T] polymorphism created by bisulfite conversion of non-CpG cytosines in the genome. These controls use Infinium I probe design and allele-specific single base extension to monitor efficiency of bisulfite conversion. If the bisulfite conversion reaction was successful, the "C" (Converted) probes matches the converted sequence and get extended. If the sample has unconverted DNA, the "U" (Unconverted) probes get extended. There are no underlying C bases in the primer landing sites, except for the query site itself. The calculation is done in both the green and red channels separately to provide 2 unique sets of values: Green Channel Lowest value of C1 or C2 / Highest value of U1 or U2. The default threshold is 1. This value can be increased for some scanners. Background/(U1, or U2). The default threshold is 1. Do not change the default threshold; however, the offset correction can be changed. Red Channel Lowest value of C3, 4, or 5 / Highest value of U3, 4, or 5. The default threshold is 1. This value can be increased for some scanners. Background /(Highest value of U4, U5, or U6). The default threshold is 1. Do not change the default threshold; however, the offset correction can be changed.
bisulfite_conversion2 bisulfite_conversion2_background	These controls assess the efficiency of bisulfite conversion of the genomic DNA. The Infinium Methylation probes query a [C/T] polymorphism created by bisulfite conversion of non-CpG cytosines in the genome. These controls use Infinium II probe design and single base extension to monitor efficiency of bisulfite conversion. If the bisulfite conversion reaction was successful, the "A" base gets incorporated and the probe has intensity in the red channel. If the sample has unconverted DNA, the "G" base gets incorporated across the unconverted cytosine, and the probe has elevated signal in the green channel. The calculation is done using both channels for 1 set of numbers returned. The following metrics are provided: (Lowest of red C 1, 2, 3, or 4) / (Highest of green C 1, 2, 3, or 4). The default threshold is 1. This value can be increased for some scanners. Background/(Highest C1, C2, C3, or C4 green). The default threshold is 1. Do not change the default threshold; however, the offset correction can be changed.
specificity1_green specificity1_red	Specificity controls are designed to monitor potential nonspecific primer extension for Infinium I and Infinium II assay probes. Specificity controls are designed against nonpolymorphic T sites. These controls are designed to monitor allele-specific extension for Infinium I probes. The methylation status of a particular cytosine is carried out following bisulfite treatment of DNA by using query probes for unmethylated and methylated state of each CpG locus. In assay oligo design, the A/T match corresponds to the unmethylated status of the interrogated C, and G/C match corresponds to the methylated status of C. G/T mismatch controls check for nonspecific detection of methylation signal over unmethylated background. PM controls correspond to A/T perfect match and give high signal. MM controls correspond to G/T mismatch and give low signal. The metrics provided are the ratio of the lowest PM/highest MM in each channel. The default threshold is 1. Do not change the default threshold.
specificity2 specificity2_background	Specificity controls are designed to monitor potential nonspecific primer extension for Infinium I and Infinium II assay probes. Specificity controls are designed against nonpolymorphic T sites. These controls are designed to monitor extension specificity for Infinium II probes and check for potential nonspecific detection of methylation signal over unmethylated background. Specificity II probes incorporate the "A" base across the nonpolymorphic T and have intensity in the Red channel. If there was nonspecific incorporation of the "G" base, the probe has elevated signal in the Green channel. The following metrics are provided: (Lowest intensity of S1, S2, or S3 red) / (Highest intensity of S1, S2, or S3 green). The default threshold is 1. Do not change the default threshold. Background/(Highest intensity S1, S2, S3, or S4 green). The default threshold is 1. Do not change the default threshold; however, the offset correction can be changed.
nonpolymorphic_green nonpolymorphic_red	Nonpolymorphic controls test the overall performance of the assay, from amplification to detection, by querying a particular base in a nonpolymorphic region of the genome. They let you compare assay performance across different samples. One nonpolymorphic control has been designed for each of the 4 nucleotides (A, T, C, and G). In the green channel, the lowest intensity of C or G is always greater than the highest intensity of A or T. The metric provided is the (lowest intensity for C or G) /(highest intensity for A or T) for a single sample. The default threshold is 5. This value can be increased for some scanners.
avg_green_raw avg_red_raw	Average green and red raw signal for the given sample.
avg_green_norm avg_red_norm	Average green and red signal after dye bias correction and noob normalization for the given sample.
ScanTime	The date (MM/DD/YY) and time (HH:MM) that the sample was scanned by the iScan system.
NProbes	Number of probes on the BeadChip, including SNP and CG probes
NPassDetection	Number of probes on the BeadChip that passed detection p-value at the threshold defined.
prop_probes_passing	The proportion of probes passing defined as the number of probes passing detection p-value divided by the total number of probes on the BeadChip.
passQC	1 = sample passed all QC metrics for the thresholds defined 0 = sample did not pass all QC metrics for the thresholds defined
failCodes	The list of parameters that failed QC for the thresholds defined.

The control metrics in the QC summary files are calculated as following. The default value for background correction offset (x) of 3,000 can be modified and applies to all background calculations indicated with (bkg + x). Note that the table uses default thresholds for EPIC arrays as example, the default thresholds changes with the methylation arrays. See section Threshold Adjustment for additional details.

Control	Calculation	Additional Information
Restoration Green > bkg	(Green/(bkg+x))>	If using the FFPE Restore kit, change the default threshold from 0 to 1. bkg = Extension Green highest A or T intensity
Staining Green Biotin High > Biotin Bkg	(High/Biotin Bkg) > 5
Staining Red DNP High > DNP Bkg	(High/DNP Bkg) > 5
Extension Green Lowest CG/Highest AT	(C or G/A or T) > 5	Green channel—Lowest C or G intensity is used; highest A or T intensity is used.
Extension Red Lowest AT/Highest CG	(A or T/C or G) > 5	Red channel—Lowest A or T intensity is used; highest C or G intensity is used.
Hybridization Green High > Medium > Low	(High/Med) > 1 (Med/Low) > 1
Target Removal Green ctrl 1 ≤ bkg	((bkg + x)/ctrl) > 1	bkg = Extension Green highest A or T intensity
Target Removal Green ctrl 2 ≤ bkg	((bkg + x)/ctrl) > 1	bkg = Extension Green highest A or T intensity
Bisulfite Conversion I Green C1, 2 > U1, 2	(C/U) > 1	Lowest C intensity is used. Highest U intensity is used.
Bisulfite Conversion I Green U ≤ bkg	((bkg + x)/U) >	For MSA arrays, the default is 0.5 Highest U intensity is used. Green channel—bkg = Extension Green highest AT
Bisulfite Conversion I Red C3, 4, 5 > U3, 4, 5	(C/U) >1	Lowest C intensity is used. Highest U intensity is used.
Bisulfite Conversion I Red U ≤ bkg	((bkg + x)/U) >	For MSA arrays, the default is 0.5 Highest U intensity is used. Red Channel—bkg = Extension Red highest CG
Bisulfite Conversion II C Red > C Green	(C Red/ C Green) >	For MSA arrays, the default is 0.5 Lowest C Red intensity is used. Highest C Green intensity is used.
Bisulfite Conversion II C green ≤ bkg	((bkg + x)/C Green) >	For MSA arrays, the default is 0.5 Highest C Green intensity is used. Green channel—bkg = Extension Green highest AT
Specificity I Green PM > MM	(PM/MM) > 1	Lowest PM intensity is used. Highest MM intensity is used
Specificity I Red PM > MM	(PM/MM) > 1	Lowest PM intensity is used. Highest MM intensity is used
Specificity II S Red > S Green	(S Red/ S Green) > 1	Lowest S Red intensity is used. Highest S Green intensity is used.
Specificity II S Green ≤ bkg	((bkg + x)/ S green) > 1	bkg = Extension Green highest A or T intensity Highest S Green intensity is used.
Nonpolymorphic Green Lowest CG/ Highest AT	(C or G/ A or T) >	Lowest C or G intensity is used; highest A or T intensity is used For MSA arrays, the default threshold is 2.5
Nonpolymorphic Red Lowest AT/ Highest CG	(A or T/ C or G) >	Lowest A or T intensity is used; highest C or G intensity is used For MSA arrays, the default threshold is 3

Methylation Sample QC Summary Plots

The software produces methylation sample QC summary plots (sample_qc_summary.pdf) per analysis batch which provides visual depictions of two QC summary plots for quick visual review.

The file contains the following control plots:

Control Plot	Description
Proportion of Probes Passing Threshold	Histogram of the proportion of probes passing the p-value detection threshold. Samples passing QC are shown in one color, and samples failing QC are shown in another color.
Principal Component Analysis (PCA)	Uses beta values for all analytical probes to compare samples. Principal component analysis (PCA) is applied to the beta values to reduce the dimensionality of the data to two “principal components” that reflect the most variation across samples. If more than 100 samples are used in the analysis, a random subset of 10,000 probes are used for the PCA analysis to reduce computational burden. PCA control plot assigns unique colors to each sample group defined by the IDAT Sample Sheet. If no groups were assigned, all samples will appear the same color. Sample groups may cluster together and can be used to explain some of the variation. Coordinates used to plot each sample in the PCA control plot are provided in the pcs.tsv.gz output file (see below).

Methylation Principal Component Summary

The software produces a methylation principal component summary file (pcs.tsv.gz) per analysis batch which provides principal component data for each sample within the batch. This can be used to identify the specific samples associated with points on the PCA control plot within the Methylation Sample QC Control Plots output file.

The files contain the following fields:

Field	Description
blank	BeadChip Barcode and Position ie 123456789101_R01C01
principal component 1	The variable of the first axis for the Principal Component Analysis
principal component 2	The variable of the second axis for the Principal Component Analysis
Sample_Group	Sample group defined by the user in the IDAT Sample Sheet. If no sample group was defined, all samples will show NA.

Methylation Manifest Files

The software produces two methylation manifest files

1. Manifest in Sesame format (probes.csv)

2. Additional information for control probes (controls.csv)

The probes.csv file has the following columns:

Field	Description
Probe_ID	This is a unique identifier for each probe. It corresponds to the IlmnID column in the standard Illumina manifest format or ctl_[AddressA_ID] for control probes.
U	This is corresponds to the AddressA_ID column in the standard Illumina manifest format.
M	This corresponds to the AddressB_ID column in the standard Illumina manifest format.
col	This is the color channel for Infinium I probes (R/G). For Infinium I probes, this column will be NA.

The controls.csv file has the following columns:

Field	Description
Address	The address of the probe
Type	The control probe type
Color_Channel	A color used to denote certain control probes in legacy software
Name	A human readable identifier for certain control probes
Probe_ID	This is a unique identifier for each probe. It corresponds to the IlmnID column in the standard Illumina manifest format or ctl_[AddressA_ID] for control probes.

Methylation Warning/Error Messages and Logs

The following scenarios result in a warning or error message:

• Missing IDATs or manifest

• Incorrect sample sheet formatting

• Duplicate BeadChip Barcode and Position within the sample sheet

• Missing control or assay probes

• Missing required columns in the manifest

• Unable to compute certain metrics

Examples of such notifications can include the following:

Log	Error	Type	Cause
write_samplesheet.log	No IDATs found	Error	No IDATs provided for analysis
format_samplesheet.log	No samples in sample sheet	Error	No samples in user’s sample sheet input
format_samplesheet.log	Sample sheet not correctly formatted	Error	Sample sheet is not in CSV format or header lines do not start with “<”
format_samplesheet.log	beadChipName and sampleSectionName columns are required for the sample sheet.	Error	Sample sheet does not contain required columns: beadChipName and sampleSectionName.
format_samplesheet.log	Warning: <Number> samples have duplicate Sample_ID	Warning	X lines in the sample sheet have duplicate <beadChipName>_<sampleSectionName>. Duplicates are dropped from analysis.
convert_manifest_ilmn_sesame.log	Missing control probes in manifest	Error	Missing “[Controls]” line in CSV manifest
convert_manifest_ilmn_sesame.log	Probe section not found	Error	Missing “[Assay]” line in CSV manifest
convert_manifest_ilmn_sesame.log	Missing required columns: IlmnID, AddressA_ID, AddressB_ID, Color_Channel	Error	Missing one of required columns in Assay section of manifest
convert_manifest_ilmn_sesame.log	Controls not formatted correctly. Must have 4 columns (Address,Type,Color_Channel,Name)	Error	Missing one of required columns in Control section of manifest
run_sesame_gs.log	Missing sample: <Sample_ID>	Error	Missing idats for a particular sample
run_sesame_gs.log	No scan time available	Warning	No scan time in idat
run_sesame_gs.log	Prep failed	Error	Dye bias correction or noob failure for sample
run_sesame_gs.log	Warning: missing control probe types <Missing probes>	Warning	Missing control probe types to compute a BACR metric. Metric will be set to NA.
run_sesame_gs.log	Warning: missing control probe names <Missing probe types>	Warning	Missing control probes to compute a BACR metric. Metric will be set to NA.
qc.log	No features, skipping PCA plot	Warning	No common betas found in all samples. This may occur if a sample has no signal intensity in the IDAT files.