Nimblegen seqcap ez human exome library v2

Tumor and blood samples from the JWH data set were obtained from patients in the practice of the senior author who were diagnosed with UM arising from the choroid and/or ciliary body, and treated by primary enucleation without previous radiotherapy. The study was approved by the Institutional Review Board at the University of Miami and written informed consent was obtained from each patient. Clinical and histopathologic information were obtained and de-identified for further analyses. WES was performed on 40 tumor samples, 21 of which had matched blood DNA samples available for sequencing. DNA was extracted using the Wizard Genomic DNA Purification kit (Promega, Madison, WI) and the Quick Gene DNA whole blood kit S (Fugifilm, Tokyo, Japan), respectively. Exome fragments were captured using NimbleGen SeqCap EZ Human Exome Library v2.0 (Roche Nimblegen) and sequenced on the Illumina Genome Analyzer II (GAIIx). RNA from these cases was isolated using the PicoPure kit and sent to Castle BioSciences, Inc. for GEP to determine class 1 versus class 2 status⁴⁷ .

Genomic DNA was extracted from blood samples using standard phenol-chloroform extraction method [9 (link)]. Exome capture was performed in the proband (III:4), by BGI-Shenzhen using NimbleGen SeqCap EZ Human Exome Library v2.0 (Roche NimbleGen, Inc., Madison, WI, USA), and sequencing was performed using a HiSeq 2000 platform (Illumina, San Diego, CA, USA). All steps were performed according to the manufacturer’s instructions [10 (link)]. The enriched library targeting the exome was sequenced on the HiSeq 2000 platform to get paired-end reads with a read length of 90-bp [11 (link)]. Exome sequencing depth of 68.01× were obtained to provide sufficient depth to accurately call variants at 99.17% of each targeted exome.

DNA samples from individuals with OM from 28 Minnesota, 217 Finnish, and 14 Pakistani families were submitted for exome sequencing using an Illumina HiSeq instrument, at an average 40–60 × coverage. For the Minnesota and Finnish families, sequence capture was performed at the University of Washington using the Roche NimbleGen SeqCap EZ Human Exome Library v.2.0. For the Pakistani families, exome sequencing was performed at the University of Maryland and genomic libraries were recovered for exome enrichment using the Agilent SureSelect Human Expanded All Exon V5 (62 Mb) kit. For all exome data, alignment and variant calling were performed using Burrows-Wheeler Aligner^{49 (link)}and Genome Analysis Toolkit^{50 (link)}, respectively.
Sanger sequencing was used to confirm co-segregation of five PLG variants (NM_00301.3) identified in exome data from nine multi-ethnic families (Table 1). An additional 70 Coloradan trios, 246 Texan trios, 1 Finnish family, 2 Minnesota families, and 1 Pakistani family without exome data were Sanger-sequenced for the PLG variants.

For whole-exome sequencing, 1 μg of DNA from two family members with episodic pain was fragmented using sonication technology (Covaris, Woburn, MA, USA). The fragments were end-repaired and adaptor-ligated including incorporation of sample index barcodes. After size selection, the libraries were subjected to an enrichment process (NimbleGen SeqCap EZ Human Exome Library v2.0, Roche NimbleGen). Samples were sequenced on an Illumina HiSeq 2000 sequencing instrument. This resulted in 6.6 (individual 1) and 6.4 (individual 2) Gb of mapped sequences with a mean coverage of 77/78 and a 30 × coverage of 78/84% and a 10 × coverage of 95.8/96.4% of target sequences. For data analysis, the Varbank pipeline v.2.3 and interface was used (https://anubis.ccg.uni-koeln.de/varbank/). Primary data were filtered according to signal purity with the illumina real-time analysis software v1.8. Subsequently, the reads were mapped to the human genome reference build hg19 using the Burrows-Wheeler alignment algorithm. GATK v.1.6 was used to mark duplicated reads, to perform a local realignment around short insertion and deletions, to recalibrate the base quality scores, and to call single nucleotide polymorphisms and short indels. Subsequent Sanger sequencing of SCN11A exons was performed using standard procedures. Primer sequences are available on request.

Genomic DNA from the index case was subject to whole exome sequencing (Welcome Trust Centre for Human Genetics, Oxford, UK) funded by Oxford NIHR Biomedical Research Centre Genomic Medicine Theme. Exome library was captured from 3 µg of genomic DNA using Roche Nimblegen SeqCap EZ Human Exome Library v2.0. The libraries were sequenced by 100 nt paired-end reads on the Illumina HiSeq platform. The obtained sequences were mapped to the human genome build hg19 by using Novoalign software (Novocraft Technologies). Variants were called using the Samtools program. We used ANNOVAR software to annotate and separate non-synonymous substitutions, splicing mutations and mutations in 3' or 5' UTRs.