Hiseq 2000 sequencing instrument

Whole-exome sequencing was performed on 16 fresh frozen atypical carcinoids in the Cologne Centre for Genomics. Exomes were prepared by fragmenting 1 μg of DNA using sonication technology (Bioruptor, Diagenode, Liège, Belgium) followed by end repair and adapter ligation including incorporation of Illumina TruSeq index barcodes on a Biomek FX laboratory automation workstation from Beckman Coulter (Beckman Coulter, Brea, CA, USA). After size selection and quantification, pools of five libraries each were subjected to enrichment using the SeqCap EZ v2 Library kit from NimbleGen (44Mb). After validation (2200 TapeStation; Agilent Technologies, CA, USA), the pools were quantified using the KAPA Library Quantification kit (Peqlab, Erlangen, Germany) and the 7900HT Sequence Detection System (Applied Biosystems, Waltham, MA, USA), and subsequently sequenced on an Illumina HiSeq 2000 sequencing instrument using a paired-end 2 × 100 bp protocol and an allocation of one pool with 5 exomes/lane. The expected average coverage was approximately 120x after removal of duplicates (11 GB).

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.

WES was performed by first fragmenting 1 μg of DNA (Bioruptor, diagenode, Liége, Belgium). The DNA fragments were then end-repaired and adaptor-ligated with sample index barcodes. Following size selection, the SeqCap EZ Human Exome Library version 2.0 kit (Roche NimbleGen, Madison, WI, USA) was used to enrich for the whole exome. The DNA libraries were then sequenced with a paired-end 2 × 100 bp protocol aiming for an average coverage of 90× and 120× for the normal and tumor DNA, respectively. The primary data were filtered for signal purity with the Illumina Realtime Analysis software.
WGS was performed with a read length of 2 × 100 bp. The samples were processed to provide 110 Gb of sequence, thus amounting to a mean coverage of 30× for both tumor and matched normal.
For RNA-seq, cDNA libraries were prepared from PolyA + RNA following the Illumina TruSeq protocol for mRNA (Illumina, San Diego, CA, USA). The libraries were sequenced with a paired-end 2 × 100 bp protocol resulting in 8.5 Gb per sample, and thus in a 30× mean coverage of the annotated transcriptome.
Whole genome, whole exome and transcriptome sequencing reactions were performed on an Illumina HiSeq 2000 sequencing instrument (Illumina, San Diego, CA, USA).