Nimblegen seqcap ez exome v3

Whole-exome sequencing (WES) was performed with genomic DNA obtained from peripheral blood of Patients IV:2 and IV:14 at the National Centre for Genomic Analysis (CNAG) in Barcelona, Spain. Exome capture was performed using Nimblegen SeqCapEZ Exome V.3 (Roche) for 64 Mb according to the manufacturer’s protocol. Sequencing and bioinformatics analysis protocols are included in the Supplementary material.

Blood samples from nine family members were collected and processed for genomic DNA isolation using the MagCore® Genomic DNA Whole Blood Kit (RBC Bioscience, USA). We performed whole-exome sequencing (WES) on samples from four family members (I-2, I-3, II-1, and III-1). Whole-exome libraries were prepared using the Kapa Hyper Prep Kit (Roche, USA) according to the protocol for NimbleGen SeqCap EZ Exome v3 (Roche, USA). Paired-end 2 × 75 bp sequencing was performed on an Illumina NextSeq 500 sequencer (Illumina Inc., USA). The raw sequencing reads were aligned to the GRCh37 human reference genome using the BWA mem algorithm, version 0.7.15. PCR duplicates were identified with the MarkDuplicates tool from Picard version 2.9.2. GATK HaplotypeCaller, version 3.7, was used to detect germline single nucleotide variants (SNV) and indels. Obtained variants/indels have been annotated using Annovar program version (2018Apr16).
On the basis of the current knowledge, we have chosen 30 candidate genes previously associated with IPF: TERC, TERT, SFTPC, SFTPA1, SFTPA2, MUC5B, MUC5C, RTEL1, PARN, ABCA3, DKC1, TINF2, IL1RN, IL8, FAM13A, TLR3, HLA- DRB1, HLA- DQB1, DSP, OBFC1, MUC2, TOLLIP, ATP11A, MDGA2, MAPT, SPPL2C, DPP9, TGFB1, NAF1, and OBFC1^{7 (link)–17 (link)}. We then looked more deeply into the exonic variants of these genes.