Ion xpress template kit

Phage genomic DNA was fragmented using Ion Xpress™ Plus gDNA Fragment Library kit following the manufacturer’s protocol (Life Technologies, Foster City, CA). The fragmented DNA was collected using Pippin Prep DNA Size Selection System (Sage Science, Beverly, MA) and assessed for concentration and size distribution using a Bioanalyzer 2100 (Agilent Technologies, Mississauga, ON). The DNA fragments were then attached to the surface of Ion Sphere particles (ISPs) using an Ion Xpress Template kit (Life Technologies) according to the manufacturer’s instructions. Template-ISPs were sequenced using 316 micro-chips using an Ion Torrent Personal Genome Machine (PGM) with an Ion PGM Sequencing 400 kit (Life Technologies). The sequence reads were filtered using PGM software to remove low quality sequences, trimmed to remove adaptor sequences and the filtered sequences were assembled. The assembled genome had a coverage of 33.4×. Gaps were identified using the Lasergene® Genomics Suite of DNAStar software (DNAStar Inc., Madison, WI). The gaps were closed by PCR using primers flanking regions adjacent to the gaps and sequencing using a 3730 Genetic Analyzer (Life Technologies). The final assembled genome was manually curated for errors.

Genomic DNA was extracted from blood lymphocytes or whole blood using QIAamp and Paxgene DNA kits (QIAGEN, Germantown, MD USA), respectively. The concentration of DNA was measured using the Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Libraries were prepared using the Ion Ampliseq Library kit 2.0 (Life Technologies, Calrlsbad, CA, USA) according to the manufacturer’s protocol using 15 ng genomic DNA. Multiplex PCR was performed with Ion AmpliSeq BRCA1 and BRCA2 Community Panel (Life Technologies), which consists of 163 primer pairs in three pools and was designed to capture all BRCA1/BRCA2 coding exons and exon-intron junctions (24,143 bp). Following adaptor and barcode ligation, libraries were pooled and amplified by emulsion PCR using the OneTouch2 system and Ion Xpress template kit (Life Technologies). Ion Sphere particles were enriched using the E/S module and sequenced on PGM using a 316 chip (Life Technologies). Full screening of BRCA1 for the detection of large rearrangements using multiplex ligation-dependent probe amplification (MLPA) was performed according to the manufacturer’s protocol (MRC Holland, Amsterdam, The Netherlands).

For each sample, the gene sequences of 16S rRNA and 18S rRNA were respectively amplified using conserved bacterial primers [17 (link)] and conserved fungal primers 817F/1196R [18 (link)]. The forward and reverse primers each contained a 10-bp barcode, as shown in Additional file 1: Table S2. PCR was carried out in quadruplicate in 25-µL reaction mixtures, each containing 1 µL (10 mM) of each forward and reverse primer, 2 µL of template DNA, 2.5 µL of reaction buffer, 2.5 µL of dNTP, and 0.2 µL of Taq DNA recombinant polymerase (Takara, Otsu, Japan). Samples were denatured at 94 °C for 3 min and then amplified using 28 cycles at 94 °C for 45 s, 50 °C for 30 s, and 72 °C for 60 s. A final extension of 10 min was added at the end of the program. Negative controls (no templates) were included to check for primer or sample DNA contamination. PCR products were excised from 1% agarose gel, purified using the MinElute Gel Extraction Kit (Qiagen, Valencia, CA, USA), and quantified using a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The resulting amplicons were pooled in equimolar ratios. Sequencing was performed on an Ion Torrent™ Personal Genome Machine using the Ion Xpress Template Kit (Life Technologies, Carlsbad, CA, USA) and the Ion 314 chip (Life Technologies) following the manufacturer’s protocol.

Genomic DNA was extracted from 10-μm-thick sections of 10% neutral FFPE adenoma tissue samples using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).
Targeted gene sequencing was performed as previously described [15 (link)]. Ten nanograms of DNA was used for multiplex PCR of covered coding regions in the KCNJ5, ATP1A1, ATP2B3, and CACNA1D genes (Ion AmpliSeq panel; Life Technologies, Grand Island, NY, USA). Fragment libraries were constructed by DNA fragmentation, barcode and adaptor ligation, and library amplification using an Ion DNA Barcoding kit (Life Technologies) according to the manufacturer’s instructions. The size distribution of the DNA fragments was analyzed on an Agilent Bioanalyzer using a High Sensitivity Kit (Agilent, Santa Clara, CA, USA). Template preparation, emulsion PCR, and ion sphere particle (ISP) enrichment were performed using an Ion Xpress Template kit (Life Technologies) according to the manufacturer’s instructions. The ISPs were loaded onto a P1 chip and sequenced using an Ion P1 sequencing kit (Life Technologies).