Sureselect human all exon v6 kit

Whole‐exome sequencing was used to analyze the genetic factors of the large IPF family. The proband (II‐4), one healthy member (II‐1) and an affected member (II‐7) were chosen for the whole exome sequencing at the Novogene Bioinformatics Institute (Figure 1b). Agilent SureSelect Human All Exon V6 kits was undertaken to capture the exomes and the sequencing platform was an Illumina HiSeq X‐10. The strategies for data filtering referred to Figure 1c as our previous described (Liu & Luo, 2018).

Genomic DNA was sequenced by next‐generation sequencing (NGS), which refer to the related studies.⁵, ⁶, ⁷, ⁸ The NGS was performed by Agilent SureSelect Human All Exon V6 kits and Illumina NovaSeq 6000 sequencing platform. The paired‐end reads (PE150) were aligned to a Genome Reference Consortium Human Genome Build 37 (GRCh37)–derived alignment set including decoy sequences using the Burrows‐Wheeler Aligner (BWA). Single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number variants were called with GATK Best Practices. The sequencing data for all samples underwent standard quality control checks. It must be achieved that the average coverage depth is more than 100 X, 90.00% of the target region sequencing depth is greater than 20X, and Q30 is not less than 90%. The pathogenicity of the variants was estimated using the American College of Medical Genetics and Genomics (ACMG) guidelines. Suspected pathogenic variation was verified by Sanger sequencing with specific primers (forward primer: 5′ ‐GAGCATGTTCCGTAATCC‐3′ and reverse primer: 5′ ‐TTGTTGGGTCTCAGTTTC‐3′). This part was conducted by the Joingenome Diagnostics Co., Ltd.

Genomic DNA was prepared from peripheral blood of the patients and other all participants using a DNeasy Blood &Tissue Kit (Qiagen, Valencia, CA, U.S.A.). Genomic DNA was extracted from the peripheral blood lymphocytes of all family members by using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, U.S.A.) following the manufacturer’s instruction. The central part of the whole exome sequencing was provided by the Novogene Bioinformatics Institute (Beijing, China). The exomes were captured using Agilent SureSelect Human All Exon V6 kits, and high-throughput sequencing was performed using Illumina HiSeq X-10. The necessary bioinformatics analyses, including reads, mapping, variant detection, filtering, and annotation, were also endowed by Novogene Bioinformatics Institute [9 (link)].
The strategies of data filtering refer to our previous study [9 (link)]: (a) variants within intergenic, intronic, and UTR regions as well as synonymous mutations were excluded for later analysis; (b) variants with MAF>0.01 in the 1000 Genomes project, dbSNP132 were excluded; (c) variants with MAF>0.01 in genome aggregation database (gnomAD) (http://gnomad.broadinstitute.org/) were further precluded; (d) SIFT, Polyphen-2 and MutationTaster were utilized to predict the possible impacts of variants. (e) Co-segregation analysis was conducted in the family.

The main part of the whole exome sequencing (WES) was provided by the Berry Genomics Bioinformatics Institute (Beijing, China). Exomes were captured using Agilent SureSelect Human All Exon V6 kits, and the platform of high-throughput sequencing was performed on an Illumina HiSeq X-10. Basic bioinformatics analysis, including Reads, Mapping, Variant detection, Filtering, and Annotation, was also performed by the Berry Genomics Bioinformatics Institute.

The genomic DNA was extracted by DNeasy blood and tissue kit (QIAGEN # 69506). And the exome capture, high throughput sequencing, and common filtering were performed in the Novogene Bioinformatics Institute (Beijing, China). All the exomes were captured by Agilent SureSelect Human All Exon V6 kits and sequenced by Illumina HiSeq X-10 platform. The strategies of data filtering are as follows as we have described [15 (link), 16 (link)]: (1) variants in the 1000 Genomes Project (1000G, http://www.1000genomes.org) with MAF > 0.01 were excluded. (2) Variants in the dbSNP144 (https://www.ncbi.nlm.nih.gov/projects/SNP/) with MAF > 0.01 were also excluded. (3) The remaining data were filtered by PCD-related genes (Table S1). (4) Bioinformatics analysis was used for the remaining variants.