Truseq sbs sequencing kit version 3

RNA sequencing and bioinformatics analysis was conducted in collaboration with the Mayo Medical Genomics Facility as previously described [12 (link)]. Quality of the RNA was assessed by the Mayo Gene Expression Core using Agilent Bioanalyzers. All samples had RNA Integrity Numbers greater than 7.0. RNA sequencing was performed as paired-end base reads on an Illumina HiSeq 2000 with three samples per lane, using the TruSeq SBS Sequencing Kit, Version 3. Base calling was performed using Illumina’s RTA version 1.12.4.2. Bioinformatics were performed with the assistance of the Mayo Division of Biostatistics and Informatics. Analysis of each sample (alignment statistics, in-depth quality control metrics, and gene and exon expression levels) was done using Mayo Clinic’s MAPRSeq v1.2. Reads were mapped using Tophat version 2.0.6 against the hg19 reference genome and gene counts were produced using htseq. Differential expression analyses between samples were computed using an edgeR version 3.3.8 algorithm. Data was deposited in the NCBI Gene Expression Omnibus (accession number GSE86007).

RNA libraries were prepared according to the manufacturer’s instructions for the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA). The concentration and size distribution of the libraries were determined on an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA, USA). Libraries were loaded onto flow cells at concentrations of 8–10 pM to generate cluster densities of 700,000/mm² following Illumina’s standard protocol using the Illumina cBot and cBot Paired End cluster kit version 3. The flow cells were sequenced as 51 × 2 Paired End reads on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 3 and SCS version 1.4.8 data collection software. Base calling was performed using Illumina’s RTA version 1.12.4.2. There were approximately 45 million reads per sample mapped to the human genome, and 21,686 genes were detected.

Total RNA was isolated as described above and RNA libraries were prepared according to the manufacturer's instructions for the TruSeq RNA Sample Prep Kit v2 (Illumina). Libraries were loaded onto paired end flow cells following the standard protocol for the Illumina cBot and cBot Paired end cluster kit version 3. Flow cells were sequenced as 51 × 2 paired end reads on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 3 and HCS v2.0.12 data collection software. Base-calling was performed using Illumina's RTA version 1.17.21.3. The RNA-Seq data was analyzed using MAP-RSeq v.1.2.1 [65 (link)], the Mayo Bioinformatics Core pipeline. MAP-RSeq consists of alignment with TopHat 2.0.6 [66 (link)] against the hg19 genome build and gene counts with the HTSeq software 0.5.3p9 (http://www.huber.embl.de/users/anders/HTSeq/doc/overview.html) using gene annotation files obtained from Illumina (http://cufflinks.cbcb.umd.edu/igenomes.html). Normalization and differential expression analysis were performed using edgeR 2.6.2 [67 (link)].

All samples were subjected to WGS on the Illumina HiSeq X instruments producing 150 base pair, paired-end reads to meet a goal of 30x mean coverage at the Broad Institute, RNA-seq using the Illumina TruSeq™ Stranded mRNA Sample Preparation kit on the Illumina HiSeq. 2000 or HiSeq. 2500 producing 101 base pair paired-end reads at the Broad Institute, and RRBS using the TruSeq SBS sequencing kit version 3 on the Illumina HiSeq. 2000 producing 51 base pair paired-end reads at the Mayo Clinic.
WGS data was process using the Picard Informatics Pipeline, with all data from a particular sample aggregated into a single BAM file which included all reads, all bases from all reads, and original/vendor-assigned quality scores. A pooled Variant Call Format (VCF) file using the latest version of Picard GATK software was generated and provided for each sample batch. Data for RNA-seq was analyzed using the Broad Picard Pipeline, which includes de-multiplexing and data aggregation. RRBS Data was collected using HiSeq data collection version 1.5.15.1 software, and the bases were called using Illumina’s RTA version 1.13.48.
For expanded details on library preparation, sequencing information, and all data processing and analyses including detection of genomic alterations, differential expression processing and calculation of Differentially Methylated Regions please see the Supplementary Methods.