Ion pgm 200 sequencing kit

cDNA libraries were constructed following the Ion Total RNA-Seq Kit for whole transcriptome protocol (PN 4467091 Rev. C (10/2011)) from Ion Torrent (Life Technologies, Carlsbad, CA, USA). Yield and quality of the libraries were assessed using an Agilent 2100 Bioanalyzer with an Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA) and cDNA were stored at -20°C. The templates were prepared following the Ion OneTouch 200 Template Kit v.2.0 protocol (PN 4478371 Rev. A (06/2012)), and stored at 4°C. Sequencing runs were performed on the Personal Genome Machine following the Ion PGM 200 Sequencing Kit protocol (PN 4474246 Rev. D (06/2012)), using Ion 316 Chip Kit, provided by Life Technologies.

A fragment library was generated with an input of 100 ng DNA using the Ion Xpress Plus Fragment Library Kit (Life Technologies, Carlsbad, USA). Next, DNA fragmentation, adaptor ligation, fragment size selection and library amplification were carried out for a total of 8 cycles according to manufacturer's instructions. The targeting of fragments of approximately 330 bp was performed with 2% agarose gel cassettes for the Pippin Prep instrument (Sage Science, Beverly, USA). Prior to emulsion PCR, the size distribution and concentrations of the library were assessed with a Bioanalyzer 2100 using the DNA High Sensitivity Kit (Agilent, Santa Clara, CA). The fragment library was adjusted to approximately 26 pM and amplified with Ion Sphere particles™ (ISPs) by emulsion PCR using the Ion OneTouch™ Instrument with the Ion OneTouch™ 200 Template Kit (Life Technologies, Grand Island, NY) and template-positive ISP enrichment according to the manufacturer's protocol (Part Number 4472430 Rev. E, 06/2012). Over 50% of the ISPs were and then sequenced on an Ion 316™ chip using the Ion Torrent Personal Genome Machine (PGM™) (Life Technologies, San Francisco, CA, USA) for 130 cycles (520 flows) with the Ion PGM™ 200 Sequencing Kit (Part Number 4474246 Rev. D, 06/2012).

Amplicon libraries were prepared using an Ion AmpliSeq Custom Panel (Applied Biosystems, Life Technologies), in accordance with the manufacturer’s instructions, for 68 genes reported to cause non-syndromic hereditary HL (Supplementary Table 1). The detailed sample preparation protocol has been described elsewhere^{12 (link),13 (link)}. Sequencing was performed in accordance with the manufacturer’s instructions. Massively Parallel Sequencing (MPS) was performed with an Ion Torrent Personal Genome Machine (PGM) system or Ion Proton System using an Ion PGM 200 Sequencing Kit with an Ion 318 Chip (Life Technologies) or Ion HiQ Chef Kit with an Ion P1 chip. The sequence data were mapped against the human genome sequence (build GRCh37/ hg19) with a Torrent Mapping Alignment Program. After sequence mapping, the DNA variants were detected with Torrent Variant Caller plug-in software. After variant detection, their effects were analyzed using ANNOVAR software^{14 (link)}.

RB733 genomic DNA was purified by phenol and chloroform extraction including
RNase A treatment. 1 μg DNA was subjected to
fragmentation and adaptor ligation using an Ion Xpress Plus Fragment Library Kit
(Life Technologies), according to the manufacturer’s instructions.
The library was subjected to emulsion PCR using the Ion OneTouch 200 Template
Kit v2 DL (Life Technologies), followed by bead enrichment. Then, whole-genome
sequencing was performed with an Ion Torrent PGM system using the Ion PGM 200
Sequencing Kit and Ion 318 chip (Life Technologies). Using IGV (Integrative
Genomics Viewer), we inspected the genome region where no read was aligned as
the potential deletion candidates. Except for ok498, we found two
potential deletions larger than 1 kb on chromosome X. One of the two
genomic regions contains no protein-coding gene, thus we focused on another
region, around chromosome X: +6.73 cM. This mutation candidate was
confirmed by Sanger sequencing and revealed a 7809 bp deletion and
7 bp insertion located in the genomic region of rsd-3.

15.0 mL of sludge obtained from the bioreactor digesting freeze dried Spirulina were used for DNA extraction (sampling day 336, biogas production, 1676 mL biogas day⁻¹, methane content 60 %). DNA was extracted according to Zhou et al. [53 (link)] with minor modifications. 2.5 μg of extracted DNA were used to prepare a 200 bp insert size sequencing library for the Ion Torrent Personal Genome Machine (PGM) platform (Life Technologies, USA). The instructions according to the Ion Xpress™ - Plus gDNA Fragment Library Preparation manual were followed, except for the initial DNA fragmentation, which was done using a GS FLX Standard Nebulizer Kit (Roche Applied Science, Germany), nebulization for 3 min at 32 psi. Sequencing template preparation was performed using the OneTouch Instrument and the OneTouch ES module. Enriched ISP particles were sequenced with the Ion PGM™ 200 Sequencing Kit (Life Technologies, USA) on a 318™ Chip with 520 flows following the manufacturer’s instructions. Automated analysis was performed with the Torrent Suite™ Software v3.2 using default settings. Additional quality filtering was done using the Trimmomatic tool v3 (http://www.usadellab.org/cms/index.php?page=trimmomatic) [54 (link)], with settings for removal of trailing bases of a q-value lower than 20, and removal of reads shorter than 50 bases.

DNA preparation and sequencing was performed at San Valley Biotechnology Incorporated, Beijing, China. Total DNA was extracted from each formalin-fixed, paraffin-embedded (FFPE) specimen using the QIAamp DNA Mini Kit (QIAGEN). An Ion Torrent adapter-ligated library was constructed using the Ion AmpliSeq Library Kit 2.0 (Life Technologies) and purified using AMPure beads (Beckman Coulter). Emulsion PCR was performed using an IKADT-20 mixer (Life Technologies) after adding Ion Sphere Particles (ISPs). The enrichment of template-positive ISPs was performed using Dynabeads MyOne Streptavidin C1 beads (Life Technologies) and confirmed using a Qubit 2.0 fluorometer (Life Technologies). Sequencing reactions were subsequently performed using the Ion PGM 200 Sequencing Kit (Life Technologies). Forty-six mutations, including EGFR, BRAF, TP53, ALK and other cancer-related gene mutations, were targeted in this study (Table 3).