Truseq dna exome kit

Whole-exome sequencing (WES) was performed on genomic DNA obtained from the patient’s peripheral blood as previously described [15 (link)]. The DNA library was prepared using the Illumina’s TruSeq DNA Exome Kit. Sequencing was performed on the NextSeq-500 platform using the NextSeq 550 High Output kit (150 cycles) according to the manufacturer protocol (Illumina, California, USA). Raw sequence reads were aligned to the human genome reference sequence hg19 using FastQC 0.11.7 and BWA (Aligner) 0.7.15. Imported variants were annotated, filtered, and classified using VariantStudio (Illumina, California, USA). The variants were further filtered using the dbSNP (https://www.ncbi.nlm.nih.gov/snp/) and the Genome Aggregation Database (https://gnomad.broadinstitute.org/). Sanger sequencing was performed to confirm the presence of the NGS-derived variant in the patient. Familial segregation analysis by Sanger sequencing was limited to the patient’s non-affected brother due to the unavailability of parents or other family members.

The family pedigree depicted cases of consanguinity but no clear pattern of inheritance. Thus for exome sequencing, affected subjects from distant branches of pedigree were chosen. Detail of sample selection criteria for exome sequencing has been explained in supplementary. DNA was isolated from blood using Qiagen DNA blood mini kit. Eight subjects- II:5, III:7, III:9, III:10, IV:31, IV:34, V:27, V:42 (five cases and three controls) were selected for exome sequencing using the Illumina TruSeq DNA exome kit for the capture of the exonic region as per manufacturer’s protocol. 6 samples were multiplexed and loaded on a single lane of the Hiseq. 2000 flow cell for sequencing. Post sequencing, data was subjected to quality check (QC) and bioinformatics analysis^{21 (link)} with minor modifications as described in Supplementary Methods (Supplementary Fig. E1A).

WES of DNA was performed at the Genomics Platform of the Broad Institute of Harvard and MIT as described previously^{67 (link),68 (link)}, with the exception of samples previously sequenced at Johns Hopkins^{65 (link)} and Yale University^{69 (link)}. In brief, DNA was extracted from FFPE tumor specimens and either matched normal whole blood, or in cases where this was unavailable, from adjacent normal FFPE specimens. Extraction was performed using the Qiagen AllPrep DNA/RNA Mini Kit (80204). A single aliquot of 150–500 ng input DNA in 100 μl TE buffer was used for library generation. Library preparation was performed using the Kapa HyperPrep kit, and quantification was performed using PicoGreen. Adapter ligation was performed using the TruSeq DNA exome kit from Illumina per manufacturer’s instructions. Sequencing of pooled libraries was performed using a HiSeq2500 with 76 bp paired-end reads. The mean target coverages for tumor and normal samples were 150× and 80×, respectively.

DNAs were extracted from tumor tissues and healthy tissues in the liver, kidney, etc. Illumina TruSeq DNA Exome kit was used for exon capture. Sequencing was carried out using Illumina 2 × 100 bp paired-end sequencing on a HiSeq 2500 instrument according to the manufacturer’s recommendation.

WES is a widely used next-generation sequencing (NGS) method that involves sequencing the protein-coding regions of the genome. We selected 15 samples of participants (8 cases and 7 controls) extracted from peripheral blood leukocytes by using the MagPurix DNA extraction kit, processed according to the TruSeq DNA Exome Kit (Illumina, San Diego, CA, USA) guidelines, and fragmented 100 ng of genomic DNA into 150 bp inserts by the M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA). Briefly, fragment gDNA is ligated with adapters and enriched by PCR. After PCR amplification, the probe captured and amplified fragments to create the WES library. The Nextseq 550 (Illumina) was used for WES. Then, those sequenced reads were aligned to the human reference genome (hg38) by using NGS Core Tools/FASTQ mapping with CLC Genome Workbench 23.0.4 (Qiagen, Hilden, Germany). We filtered out and compared those detected variants with the SNPs database (p < 0.05). SNPs profiles were assessed by using the resequencing analysis/variant comparison module of CLCs. The atlas of NCBI HPA RNAseq was utilized to compare infertile men (experimental data) to fertile men (atlas), and our team noticed specific genes that were shown to have a significant expression in testicular tissues in infertile men.

Genomic DNA was extracted from flash-frozen tumors using the QIAGEN DNeasy Blood & Tissue Kit. Sequencing libraries were prepared using the Illumina TruSeq DNA Exome kit, prior to pooling in groups of 12 and sequencing using a NextSeq 550 (Illumina) with a NextSeq 500/550 High Output Kit v2.5 (150 cycles) using paired-end settings (2 × 75).