Exome libraries captured using the SureSelect Human All Exon V4 or V5 kit (Agilent, Santa Clara, CA, USA) for WES were sequenced on an Illumina platform. Most bioinformatic analyses of sequencing data were performed using the computing server at the Genomic Medicine Institute Research Service Center. We aligned the DNA sequence reads to the human reference genome (GRCh37) or a combined reference genome of human (GRCh37) and mouse (mm10) using Burrows–Wheeler Aligner (BWA) [11 (link)]. Sorting of reads and marking of PCR-duplicated reads were performed using Picard (http://broadinstitute.github.io/picard/ ). For samples using a combined reference genome of human and mouse, we removed mouse sequence reads according to additional processing procedures [12 (link)]. Thereafter, we performed preprocessing procedures for bam files, including local realignment around insertions/deletions (indels) and base recalibration, according to the Genome Analysis Toolkit (GATK) best practices document [13 , 14 (link)].
Sureselect human all exon v4 or v5 kit
Manufactured by Agilent Technologies
Sourced in United States
The SureSelect Human All Exon V4 or V5 kit is a targeted sequencing solution designed for the enrichment of human exonic regions. It provides a comprehensive, cost-effective approach to capture and sequence the protein-coding regions of the human genome.
Automatically generated - may contain errors
5 protocols using sureselect human all exon v4 or v5 kit
1
Exome Sequencing Data Analysis
2
Exome Sequencing and Comprehensive CNV Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Exome targets were captured using the Agilent SureSelect Human All Exon V4 or V5 kit (Agilent Technologies, Santa Clara, CA). Raw sequencing data (FASTQ format) were generated via the Illumina HiSeq 2000 platform (Illumina, Inc., San Diego, CA). The Burrows Wheeler Alignment tool (BWA) v0.2.10 [17 (link)] was employed for sequencing data alignment to the Human Reference Genome (NCBI build 37, hg 19). All data were assessed using FastQC (version 0.11.2) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) for quality.
CNVs were generated using the following three CNV detection programs: (1) XHMM v1.0 [18 (link)], (2) CoNIFER v0.2.2 [19 ], and (3) CNVnator v0.2.7 [20 (link)]. XHMM includes several analytic steps and involves a number of parameters. In our study, we set all parameters to default (minTargetSize: 10; maxTargetSize: 10,000; minMeanTargetRD: 10; maxMeanTargetRD: 500; minMeanSampleRD: 25; maxMeanSampleRD: 20; maxSdSampleRD: 150) for filtering samples and targets, and prepared the data for normalization via XHMM. The only parameter that could be adjusted on Conifer was SVD, which was set to 1. For CNVnator, we set the bin size to 50–60 according to the average coverage depth of our sequencing data (45–70 X). XHMM and CoNIFER used a pooled sample calling approach as input, and CNVnator called CNVs sample by sample after individually generating a baseline.
CNVs were generated using the following three CNV detection programs: (1) XHMM v1.0 [18 (link)], (2) CoNIFER v0.2.2 [19 ], and (3) CNVnator v0.2.7 [20 (link)]. XHMM includes several analytic steps and involves a number of parameters. In our study, we set all parameters to default (minTargetSize: 10; maxTargetSize: 10,000; minMeanTargetRD: 10; maxMeanTargetRD: 500; minMeanSampleRD: 25; maxMeanSampleRD: 20; maxSdSampleRD: 150) for filtering samples and targets, and prepared the data for normalization via XHMM. The only parameter that could be adjusted on Conifer was SVD, which was set to 1. For CNVnator, we set the bin size to 50–60 according to the average coverage depth of our sequencing data (45–70 X). XHMM and CoNIFER used a pooled sample calling approach as input, and CNVnator called CNVs sample by sample after individually generating a baseline.
3
Exome Sequencing of KIF1A Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was obtained from peripheral blood leukocytes using Quick-Gene 610L (Wako, Osaka, Japan). Genomic DNA was captured using the SureSelect Human All Exon v4 or v5 Kit (51 Mb; Agilent Technologies, Santa Clara, CA, USA) and sequenced on a HiSeq2000 (Illumina, San Diego, CA, USA) with 101-bp paired-end reads. Exome data processing, variant calling and variant annotation were performed as described previously. 10 Rare nonsynonymous KIF1A variants, which were absent in 137, the 6500 exomes of the National Heart, Lung and Blood Institute exome project, and our in-house 575 control exomes, were considered as candidate KIF1A mutations, and their segregation was examined by Sanger sequencing with trio samples (patients and their parents). In families showing de novo mutations, parentage was confirmed by microsatellite analysis, as previously described. 11 Pathogenicity of the mutations was predicted using Sorting Intolerant from Tolerant (SIFT; http://sift.jcvi.org/), Polyphen2 (http://genetics.bwh.harvard.edu/pph2/) and Mutation Taster (http://www.mutationtaster.org/). KIF1A mutations were annotated based on transcript variant 1 (NM_001244008.1). The de novo KIF1A mutations were deposited to a gene-specific database (http://databases.lovd.nl/ shared/genes/KIF1A).
4
Comprehensive Tumor Genomic Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For primary bulk tumor analysis, a total of 109 sample pairs (tumor and matched normal specimens) were subjected to sequencing analysis (104 and 5 cases for CS panels and exome, respectively). DNA (200 and 1000 ng, respectively) was used for targeted re-sequencing and whole-exon sequencing. Following shearing, end repair, phosphorylation, and ligation of the barcoded adapters, DNA was subjected to hybrid capture with SureSelectXT Custom or SureSelect Human All Exon V4 or V5 kits (Agilent Technologies; the V4 and V5 kits were used before and after January 2013, respectively; Supplementary Data 2 and Supplementary Tables 2 and 3 ). The captured DNA was multiplexed and sequenced using an Illumina HiSeq2500 with a median coverage of 282–810 reads per tumor and 166–683 reads per normal specimen on the CS-A panel; 310–1936 reads per tumor and 261–1126 reads per normal specimen on the CS-B panel; and 225–354 reads per tumor and 80–197 reads per normal specimen for whole exons. The KAPA HyperPlus Kit (KAPA Biosystems) was used to construct libraries from DNA samples extracted from the FFPE tissues. When a combinatorial analysis of exome and target panel sequencing data was needed, exome data was down-sized with the merged BED file for CS-A and CS-B panels.
5
Whole-Exome Sequencing Bioinformatics Pipeline
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole‐exome sequencing (WES) was performed as described previously.7 Patient DNA was captured with SureSelect Human All Exon V4 or V5 kits (Agilent Technologies, Santa Clara, CA, U.S.A.) and sequenced on an Illumina HiSeq2000 or 2500 (Illumina, San Diego, CA, U.S.A.) with 101‐bp paired‐end reads. Image analysis and base calling were performed by sequence control software real‐time analysis and CASAVA software v1.8 (Illumina). Reads were aligned to the human reference genome sequence (UCSC hg19, NCBI build 37) using Novoalign (Novocraft Technologies, Jaya, Malaysia). Polymerase chain reaction (PCR) duplicates were excluded by Picard (http://picard.sourceforge.net/ ). Single‐nucleotide variants (SNVs) and small insertion/deletions (indels) were identified with the Genome Analysis Toolkit UnifiedGenotyper (6) and were filtered according to the Broad Institute best‐practice guidelines (version 3). Variants that were selected through the filters were annotated using ANNOVAR.8 Variant pathogenicity was evaluated by SIFT (http://sift.jcvi.org/ ), Polyphen‐2 (http://genetics.bwh.harvard.edu/pph2/ ), M‐CAP (http://bejerano.stanford.edu/mcap/ ), and CADD (http://cadd.gs.washington.edu/ ). Conservation of nucleotides was assessed with GERP (http://mendel.stanford.edu/SidowLab/downloads/GERP/index.html ) and PhastCons (http://compgen.cshl.edu/phast/ ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!