5500 solid fragment library core kit

Tissue samples were collected and homogenized with a homogenizer (Ultra-Turrax, IKA, DE) in lysis buffer RA1 of the NucleoSpin total RNA isolation kit (RNA II, Macherey-Nagel, DE). RNA isolation was performed following manufacturer’s protocol. RNA purity, concentration and integrity were determined by respectively Nanodrop (ND-1000, Isogen Life Science, NL), Qubit RNA Broad-Range (2.0, Life Technologies), and Bioanalyzer RNA Nano (2100, Agilent Technologies). Amplified double stranded cDNA was generated using the Ovation RNA-Seq system (V2, Nugen) following manufacturer’s protocol with 32 ng RNA input per sample. cDNA purity, concentration and size distribution were determined by Nanodrop, Qubit DNA HS and Bioanalyzer DNA 1000. Libraries were made using the 5500 SOLiD Fragment Library Core kit (Life technologies) and size distribution was determined by Bioanalyzer DNA 1000. RNA sequencing was performed on the SOLiD 5500 Wildfire (Life technologies). Sequence tags were mapped with Lifescope on the AllMis1 Aug. 2012 contig assembly of the American alligator genome.

DNA concentration was determined by means of fluorometric measurement (Qubit, Thermo) and quality was checked by means of determining the absence of degradation and presence of High Molecular weight DNA. Circa 1,5 μg DNA was sheared by sonication followed by barcoded adaptor ligated library construction, using the Biomek FX automated liquid handler (Beckman Coulter). Solid fragment Library preparation kit and solid barcoded adaptors were used according manufacturers protocol (Life Technologies, 5500 SOLID™ Fragment Library Core Kit, Catalog number 4464412). Each sample was generated using a separate 10 bp barcode incorporated in the adaptor sequence. After 8 cycles of amplification using the library prep kit supplied PCR primers, PCR mix and PCR protocol and purified twice-using ampure. Two rounds of hybridization capture was performed using a Custom Complement Capture (Nimblegen, pn 130204_HG19_CompCapV2_MJ_EZ_HX3). Equimolar pooling of the captured libraries was based on concentration and average sample size. Emulsion PCR was performed using the Solid EZ Bead Emulsifier and Amplifier (Applied Biosystems). Sequencing was performed on the Solid 5500xl sequencer (Life technologies) generating paired-end reads (50 bp forward and 35 bp reverse).

SOLiD sequencing libraries (rST and SZ samples) were prepared from 5caC-, 5hmC-, and 5mC-DIP enriched DNA as stated in the Lifetech Solid 5500 Chip-Seq library preparation guide. Enzymes and reagents were used from the 5500 SOLiD Fragment Library Core Kit (Life Technologies, 4464412). Fifteen cycles of library amplification were carried out using primers specific to the library sequencing adapters. Barcoded DNA fragment libraries were quantified using the Kapa Library Quantification kit (Kapa Biosystems, KK4823) and pooled equally. The Solid EZ bead system was used according to the manufacturer’s guidance to prepare ePCR and enrichment of templated beads.

The “shotgun” libraries were prepared for the sequencing platforms SOLiD 4, SOLiD 5500 and Ion Torrent (Life Technologies, USA). The sequencing was performed according to the manufacturer’s instructions. For SOLiD 4, the SOLiD Fragment Library Construction Kit, SOLiD Fragment Library Barcoding Module 1–16, SOLiD EZ Bead TM E80 System Consumables, SOLiD ToP Sequencing Kit and MM50/5 (Life Technologies, USA) were used. For SOLiD 5500, the 5500 SOLiD Fragment Library Core Kit, SOLiD Fragment Library Barcoding Kit, SOLiD FlowChip Kit, SOLiD FWD SR S50 Kit and SOLiD Run Cycle Buffer Kit (Life Technologies, USA) were used. For Ion Torrent PGM, the Ion Xpress Plus Fragment Library Kit, Ion Sequencing Kit, Ion PGM Template OT2 200 Kit, Ion PGM Sequencing 200 Kit and Ion 318 Chip Kit (Life Technologies, USA) were used.
Three of the samples were sequenced using more than one platform; in total, 28 metagenomic read sets were obtained (Additional file 1: Table S8).

Genomic DNA (gDNA) from normal liver, diaphragmatic tumour (NSCLC), lung tumour (SCLC) and liver tumour (SCLC) from patient #7 was extracted from OCT-embedded frozen tissue blocks using the DNAdvance kit from Agencourt. Three micrograms of gDNA from each sample were fragmented to approximately 150–200 bp by sonication and subjected to exome enrichment using the SureSelect^XT Human All Exon Target Enrichment system. Barcoded deep sequencing libraries for the exome-enriched gDNA fragments were constructed using Applied Biosystems SOLiD 5500 Fragment Library Core Kit. WES was performed with an Applied Biosystems SOLiD 5500 deep sequencer to generate paired-end colour space reads (50 nucleotides forward and 35 nucleotides reverse) by a multiplexed operation. The colour-space data were aligned to the human hg19 reference genome sequence by the Applied Biosystems LifeScope software to generate BAM files. Mutation calls were made using the muTect mutation calling software.