Sequencing libraries were prepared according to the TruSeq DNA v2 sample preparation EUC #15026489 revA using reagents from the TruSeq DNA v2 sample prep kit set A and set B (Illumina). A 14 pM solution of 5-6 DNA libraries pooled in equimolar amounts was subjected to cluster generation on the cBot instrument (Illumina Inc.) using the TruSeq PE cluster kit v3. Paired-end sequencing was performed for 100 cycles using a HiSeq2000 instrument (Illumina Inc.) using TruSeq SBS chemistry v3, according to the manufacturer’s protocols. Base calling was done on the instrument by RTA 1.13.48 and the resulting bcl files were filtered, demultiplexed, allowing for one mismatch base, and converted to fastq format with tools provided by CASAVA-1.8 (Illumina Inc.). Copy number profiles were computed from NGS-data using the Control-FREEC software40 (link). The sequence read depth was used to calculate an equivalent to the Log R Ratio using a sliding window approach. We applied a fixed window size of 5 kb and the other settings were default. The NGS dataset was composed of 100 whole genomes with 5x average coverage and additional exomes, with an average coverage of 17x.
Truseq sbs v3 chemistry
Manufactured by Illumina
Sourced in United States
The TruSeq SBS v3 chemistry is a sequencing-by-synthesis reagent kit developed by Illumina. The kit is designed to be used with Illumina sequencing platforms to generate high-quality sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (4 mentions)
Truseq pe cluster kit v3, by Illumina (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
Casava 1, by Illumina (2 mentions)
Truseq dna sample preparation v2, by Illumina (2 mentions)
Truseq dna sample prep kit v2 set a, by Illumina (2 mentions)
Truseq dna sample preparation kit, by Illumina (2 mentions)
Hiseq 2500 instrument, by Illumina (2 mentions)
Agilent sureselect human all exon v5 kit, by Agilent Technologies (2 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (2 mentions)
Allprep dna rna kit, by Qiagen (2 mentions)
Truseq small rna sample preparation kit, by Illumina (1 mentions)
Rta 1, by Illumina (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Cbot instrument, by Illumina (1 mentions)
Casava 1 7 or 1, by Illumina (1 mentions)
Multiplexing pe adaptors, by Illumina (1 mentions)
11 protocols using truseq sbs v3 chemistry
1
Whole Genome Sequencing and Copy Number Analysis
2
Comprehensive RNA Sequencing Protocol
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Total RNA was extracted from each tissue with the MiRNeasy kit (Qiagen, Valencia, CA, USA) according to the standard protocol. Total RNA quality was determined using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). A paired-end Illumina mRNA-Seq library was built for each tissue sample using the TruSeq RNA Sample Preparation kit (Illumina, San Diego, CA, USA) according to the standard protocol. A single-end Illumina small RNA-Seq library was built for each tissue sample using the TruSeq Small RNA Sample Preparation kit (Illumina, San Diego, CA, USA). All Illumina libraries were quantified using the standard Illumina qPCR quantification protocol. Indexed samples were multiplexed at a concentration of 1 pM each in sets of four and clustered on an Illumina cBot. Paired-end sequencing of mRNA libraries was performed for 200 cycles and single-read sequencing of small RNA libraries was performed for 50 cycles on an Illumina HiSeq 2000 instrument using TruSeq SBS chemistry v3 (manufacturer’s protocols). Base calling was done on the instrument by Illumina RTA 1.13.48. De-multiplexing and conversion of per-cycle base call files (*.bcl) to FASTQ files was done using CASAVA 1.8.2. De-multiplexing criteria allowed for one mismatch, and the resulting FASTQ files contained Sanger format ASCII encoded quality scores.
3
Whole Genome Sequencing and Copy Number Analysis
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Sequencing libraries were prepared according to the TruSeq DNA v2 sample preparation EUC #15026489 revA using reagents from the TruSeq DNA v2 sample prep kit set A and set B (Illumina). A 14 pM solution of 5-6 DNA libraries pooled in equimolar amounts was subjected to cluster generation on the cBot instrument (Illumina Inc.) using the TruSeq PE cluster kit v3. Paired-end sequencing was performed for 100 cycles using a HiSeq2000 instrument (Illumina Inc.) using TruSeq SBS chemistry v3, according to the manufacturer’s protocols. Base calling was done on the instrument by RTA 1.13.48 and the resulting bcl files were filtered, demultiplexed, allowing for one mismatch base, and converted to fastq format with tools provided by CASAVA-1.8 (Illumina Inc.). Copy number profiles were computed from NGS-data using the Control-FREEC software40 (link). The sequence read depth was used to calculate an equivalent to the Log R Ratio using a sliding window approach. We applied a fixed window size of 5 kb and the other settings were default. The NGS dataset was composed of 100 whole genomes with 5x average coverage and additional exomes, with an average coverage of 17x.
4
Exome Sequencing Library Preparation
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DNA was quantified using a Qubit Fluorometer (Life Technologies, US). Sequencing libraries were prepared according to the TruSeq DNA Sample Preparation Kit EUC 15005180 or EUC 15026489 (Illumina, US) at an average coverage of 100×. Briefly, 1–1.5 ug of genomic DNA was fragmented (Covaris, Inc., US) and all samples were subjected to end-repair, A-tailing, and adaptor ligation (Illumina Multiplexing PE adaptors). A gel-based size selection step was performed, and the adapter-ligated fragments enriched by PCR, followed by purification using Agencourt AMPure Beads (Beckman Coulter, Sweden). Exome capture was performed by pre-pooling equimolar amounts and performing enrichment in 5- or 6-plex reactions according to the TruSeq Exome Enrichment Kit Protocol (EUC 15013230). Library size was analyzed on a Bioanalyzer High Sensitivity DNA chip (Agilent Technologies, Sweden) and concentration calculated by quantitative PCR. The pooled DNA libraries were clustered on a cBot instrument (Illumina) using the TruSeq PE Cluster Kit v3. Paired-end sequencing was performed for 100 cycles using a HiSeq 2000 instrument (Illumina) with TruSeq SBS Chemistry v3, according to the manufacturer’s protocol. Basecalling was performed with RTA (1.12.4.2 or 1.13.48) and the resulting BCL files were filtered, de-multiplexed, and converted to FASTQ format using CASAVA 1.7 or 1.8 (Illumina).
5
Exome Sequencing of Tumor and Normal
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Approximately 1 mg of genomic DNA from tumor and matched normal tissue were used for library construction using the Agilent SureSelectXT Human All Exon V5 kit (covering 50 mega-bases of exonic sequence). Libraries were paired-end sequenced using Illumina’s TruSeq SBS chemistry v3 on a HiSeq2500, resulting in a median depth of coverage in the targeted regions ranging from 140 to 422 for tumor samples (median across samples: 271), and 43–233 for normal samples (median across patients: 87).
6
Exome Sequencing for Tumor Profiling
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High quality DNA was isolated using the Promega Wizard Genomic DNA Purification Kit (Promega, Wisconsin, United States) and the QIAamp DNA FFPE Tissue kit (Qiagen, Venlo, Netherlands) as previously described. One microgram of genomic DNA was used to produce exome-captured sequencing libraries using the Agilent SureSelect Human All Exon v5 kit (Agilent Technologies, California, United States). Paired-end 100-bp sequencing of each exome capture library was done using an Illumina HiSeq 2500 instrument and Illumina’s TruSeq SBS v3 chemistry (Illumina, California, United States).
Reads from tumor and matched normal blood sample were aligned separately to the human NCBI Build GRCh37 reference genome using Novoalign (Novocraft Technologies, Selangor, Malaysia) with default parameters. PCR duplicates, improper pairs and ambiguously mapped reads were removed using in-house scripts. SNVs were called using MuTect [13 (link), 14 (link)]. Variants annotation was done using Oncotator.
Reads from tumor and matched normal blood sample were aligned separately to the human NCBI Build GRCh37 reference genome using Novoalign (Novocraft Technologies, Selangor, Malaysia) with default parameters. PCR duplicates, improper pairs and ambiguously mapped reads were removed using in-house scripts. SNVs were called using MuTect [13 (link), 14 (link)]. Variants annotation was done using Oncotator.
7
Bovine Genomic DNA Sequencing from Semen
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Genomic DNA of an affected bull was prepared from a semen sample following standard protocols using proteinase K digestion and phenol-chloroform extraction. A gDNA sequencing library with 420 bp insert size was prepared using the TruSeq DNA Sample Preparation Kit (Illumina inc., San Diego, CA, USA). The sample was sequenced on an Illumina HiSeq2500 system using TruSeq SBS v3 chemistry (Illumina inc., San Diego, CA, USA) and the 2x100 bp paired-end read module. The fastq-files were generated with the CASAVA bcl2fastq conversion software (version 1.8.3, Illumina inc., San Diego, CA, USA). The alignment of the reads to the University of Maryland reference sequence (UMD3.1, [37 (link)]) was performed with the Burrows-Wheeler Aligner [41 (link)]. The resulting SAM file was converted into a BAM file with SAMtools [42 (link)]. Duplicate reads were identified and marked with the MarkDuplicates command of Picard-tools [43 ].
8
RNA Extraction and Sequencing of Human Tumor Sample
9
Exome Sequencing Library Preparation
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High quality genomic DNA was isolated using the Promega Wizard Genomic DNA Purification Kit (Promega, Wisconsin, United States) and the QIAamp DNA FFPE Tissue kit (Qiagen, Venlo, Netherlands) as previously described [47 (link)]. One microgram of genomic DNA was used to produce exome captured sequencing libraries using the Agilent SureSelect Human All Exon v5 kit (Agilent Technologies, California, United States). Paired-end 100-bp sequencing of each exome capture library was done using an Illumina HiSeq 2500 instrument and Illumina's TruSeq SBS v3 chemistry (Illumina, California, United States).
10
RNA Extraction and Sequencing of Human Tumor Sample
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RNA was extracted from a pre-treatment human fresh frozen tumor sample
for one patient with available tissue (patient #12) using AllPrep DNA/RNA kit
(Qiagen, Manchester, UK) according to manufacturer’s instructions.
Indexed PolyA libraries were prepared using 200ng of total RNA and 14 cycles of
amplification with the Agilent SureSelect Strand Specific RNA Library Prep Kit
for Illumina Sequencing (Agilent, G9691B, Santa Clara, CA, US). Libraries were
quantified by qPCR using the KAPA Library Quantification Kit for Illumina
platforms (Kapa Biosystems Inc., KK4873, Wilmington, MA, US). Paired-end 100bp
sequencing was carried out by clustering 15pM of pooled libraries on the cBot
and sequenced on the Illumina HiSeq 2500 in high output mode using TruSeq SBS V3
chemistry (Illumina Inc., San Diego, CA, US). After removing adapters using
Cutadapt (v1.14) and trimming poor quality base calls using Trimmomatic
(v0.36)49 (link), the human
reads were aligned to GRCh37 (release 75) using STAR (v2.5.1) aligner50 (link), respectively.
for one patient with available tissue (patient #12) using AllPrep DNA/RNA kit
(Qiagen, Manchester, UK) according to manufacturer’s instructions.
Indexed PolyA libraries were prepared using 200ng of total RNA and 14 cycles of
amplification with the Agilent SureSelect Strand Specific RNA Library Prep Kit
for Illumina Sequencing (Agilent, G9691B, Santa Clara, CA, US). Libraries were
quantified by qPCR using the KAPA Library Quantification Kit for Illumina
platforms (Kapa Biosystems Inc., KK4873, Wilmington, MA, US). Paired-end 100bp
sequencing was carried out by clustering 15pM of pooled libraries on the cBot
and sequenced on the Illumina HiSeq 2500 in high output mode using TruSeq SBS V3
chemistry (Illumina Inc., San Diego, CA, US). After removing adapters using
Cutadapt (v1.14) and trimming poor quality base calls using Trimmomatic
(v0.36)49 (link), the human
reads were aligned to GRCh37 (release 75) using STAR (v2.5.1) aligner50 (link), respectively.
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