SwiftBio Accel-Amplicon® BRCA1/ BRCA2 panel

Overview

Sequencing libraries were prepared from five Coriell Institute blood DNA samples using the Accel-Amplicon Plus BRCA1 / BRCA2 panel from Swift Biosciences. The libraries were sequenced using the MiSeq instrument with paired-end 150 PE reads. Each sample had one known pathogenic mutation in the BRCA1 gene. Details of the pathogenic mutations found in each patient may be found in Table I. A results summary for each patient, linked to a detailed report, is provided in the Individual Patient Report section. Details of the data processing, performance metrics of the assay, and numbers of variants identified at each filtering step are given in the Data Processing section.

Table I. Pathogenic variants identified

Coriell Sample ID Sex Age Gene Chr.Pos. Ref Alt Protein change Reference
   NA13705  F  38  BRCA1  41243789  GACA    p.S1206fs*10, p.S1253fs*10  Castilla et al., 1994(2)
   NA13715  F  56  BRCA1  41209082  GGG  GGGG  p.Q1709fs*74, p.Q1756fs*74,
p.Q1777fs*74, p.Q247fs*74,
p.Q614fs*74, p.Q652fs*74,
p.Q652fs*?, p.Q66fs*74
 Simard et al., 1994(1)
   NA14090  F  43  BRCA1  41276045  CT    p.E23fs*17  Judkins et al., 2005(3)
   NA14634  M  68  BRCA1  41243480  GATT    p.N1308fs*10, p.N1355fs*10  Simard et al., 1994(1);
Maxwell et al., 2017(4)
   NA14636  F  56  BRCA1  41197729    T  p.Y163*, p.Y1806*,
p.Y1853*, p.Y1874*,
p.Y344*, p.Y711*,
p.Y749*, p.Y86*
 Friedman et al., 1994(5);
Tung et al., 2015(6)

Table of pathogenic variants identified in each sample. Chromosomal position is given for chromosome 17 in the hg19 genome reference. Ref gives the sequence in the reference genome while Alt give the sequence found in the sample. A blank in the Ref column indicates that an insertion has been identified, whereas a blank in the Alt column indicates a deletion. Reference gives a curated list of reference(s) of interest for each variant. Protein Change is linked to the associated webpages in ClinVar database for the variant. A description of the protein naming convention can be found here.


Individual Patient Report

Click on any sample to open up a sample results sheet for this sample


Data Processing

Processing of FASTQ and BAM

Illumina adapters were removed from the 3’-end of sequence reads and low-quality bases (bases with a quality score of less than 20) were trimmed from both ends of the sequence reads using Trimmoatic v 0.36. Following Trimming the reads were aligned to the hg19 reference genome using bowtie2 v 2.2.6. Read groups were added to the .bam files using GATK AddOrReplaceReadGroups v 4.1.2.0. Primer adapters were then removed using primerclip v 0.3.8. The coverage was calculated using the merged target bed file provided by SwiftBio using bedtools coverage v 2.25.0. The percentage of the reads that were on target with the merged target bed file was calculated using GATK CountReads v 4.1.2.0. The coverage metric and percent of reads on target is shown below in table II.

Table II. Coverage and on target

Coriell Sample ID Coverage Depth 5% Coverage Depth 50% Coverage Depth 95% % of aligned reads on target
   NA13705  2052  737  53  97.22%
   NA13715  1945  695  48  96.84%
   NA14090  2031  723  41  97.05%
   NA14634  1778  610  43  91.94%
   NA14636  1992  710  41  97.00%

Coverage Depth is the minimum depth of coverage at the nucleotide level for the top n percent of nucleotides considered, as given in the Table header row. Percent (%) of aligned reads on target is the number of aligned reads that fall at least partially into the provided merged coverage areas.


Variant Calling and Analysis

The quality tables for the .bam files were recalculated using GATK BaseRecalibrator v 4.1.2.0 this was followed by variant calling using GATK HaplotypeCaller v 4.1.2.0. The .vcf files were split into SNP and Indel parts using GATK SelectVariants v 4.1.2.0 and each file was counted using GATK CountVariants v 4.1.2.0. The original .vcf file were annotated and filtered using Qiagen Ingenuity Variant Analysis (IGV) software. The found variants were sequentially filtered by first removing low confidence variants , then removing common variants , followed by removal of variants that were not predicted to be deleterious based on being absent from reference databases or if lacking a probable effect on the protein sequence. The number of retained variants at this stage are shown in the Predicted Deleterious column of Table III. The data for these remaining variants were manually examined in order to identify those which were predicted to be pathogenic by the Qiagen IVA software using guidelines from the American College of Medical Genetics(21). This classification guideline groups variants into 5 possible categories pathogenic, likely pathogenic, uncertain significance, likely benign, and benign based on Missense prediction, Splice site prediction, Nucleotide conservation prediction, Well-established functional studies, and many other factors. In all samples one pathogenic variant was identified which was known from previous sequencing of the samples found in the BRCA1 gene and reported in Table1 and 2. Sample NA13705, had two likely pathogenic variants in BRCA2 within the Predicted Deleterious Filtering category (Table III).

Table III. Overview of number of found variants.

Coriell Sample ID Total
variants
SNP
Variants
Indel
Variants
High
Confidence
Variants
Un-Common
Variants
Predicted
Deleterious
Pathogenic Variant
Frequency
   NA13705  32  25  7  23  7  6  1  43.69%
   NA13715  30  28  2  21  4  3  1  51.79%
   NA14090  27  24  3  17  4  3  1  34.58%
   NA14634  17  14  3  15  3  2  1  49.57%
   NA14636  21  17  4  20  7  4  1  41.43%

High Confidence Variants, Un-Common Variants, and Predicted Deleterious represent the number of variants after that filtering step. Pathogenic columns shows the number of variants with this classification. Variant Frequency gives the frequency of the known pathogenic variant in the sequencing data.

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