Ion pgm sequencing 200 kit v2

The LR-PCR and corrective PCR products were used to prepare libraries with an Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific Inc). In each assay batch, the LR-PCR–derived libraries and corrective PCR-derived libraries from six patients were mixed at a ratio of 7:3 to yield barcoded libraries, each of which had a total nucleotide concentration of 26 pmol/L in the mixture. After treating the samples using an Ion PGM Template OT2 Kit (Thermo Fisher Scientific Inc) according to the manufacturer’s protocol [21 (link)], emulsion PCR was performed using the Ion OneTouch 2 System (Thermo Fisher Scientific Inc), followed by enrichment of the beads using Ion OneTouch ES (Thermo Fisher Scientific Inc). The enriched emulsion-PCR products were prepared for sequencing using an Ion PGM Sequencing 200 Kit v2 (Thermo Fisher Scientific Inc) and then loaded onto an Ion 318 v2 chip (Thermo Fisher Scientific Inc) and sequenced with an Ion PGM.

Based on the qPCR quantification of libraries, equimolar amounts of each library were pulled for a final combined molarity of 400 pM. The emPCR was run by using the Ion PGM Template OT2 200 Kit (Thermo Fisher Scientific) and loaded in the Ion OneTouch™ 2 instrument. Next, non-templated Ion Sphere Particles (ISP) beads were eliminated by magnetic bead purification. The mixture of barcoded libraries was sequenced with the Ion PGM Sequencing 200 Kit V2 (Thermo Fisher Scientific). The sequencing beads were loaded in Ion 318 v2 or 316 v2 Chips (Thermo Fisher Scientific), and sequenced on the Ion Torrent™ PGM sequencer (Thermo Fisher Scientific).

The obtained barcoded clonally amplified libraries were loaded onto 318 chip and sequenced using the Ion PGM Sequencing 200 Kit v2 and IonTorrent Personal Genome Machine according to the manufacturer’s instructions (Thermo Fisher Scientific).

Genomic DNA was isolated from chorionic villi or tissue using a TIANamp Genomic DNA Kit (Beijing Tiangen Biotech Co. Ltd., Beijing, China). Genomic DNA from POCs was sheared to 250 to 300 bp fragments using an Ion Shear Plus Reagents Kit (Thermo Fisher Scientific, Waltham, MA). Ion Torrent barcoded libraries were made using an Ion Plus Fragment Library Kit (Thermo Fisher Scientific). An Ion PGM Template OT2 200 Kit (Thermo Fisher Scientific) was used for template amplification and enrichment of the target sequence. Ion sphere particles were recovered, and template-positive ion sphere particles were enriched using an Ion OneTouch ES (Thermo Fisher Scientific). Sequencing was performed using an Ion PGM Sequencing 200 Kit v2 (Thermo Fisher Scientific) on a 318 sequencing chip for a total of 500 nucleotide flows, yielding average read lengths of 220 to 230 bp. Ten DNA samples were pooled together and labeled with different barcodes on the 318 chip. The average whole genomic sequence depth was approximately 0.02×, and the average read number was approximately 500 K. The primary sequencing BAM data were submitted to the PGX cloud server (available at http://www.pgxcloud.com/), which was offered by a third-party company (JBRH, China), to analyze the chromosomal CNVs. The data analysis pipeline was established according to previous reports.^{[12 (link),13 (link)]}

A total of 107 primary tumor samples were sent for histologic assessment followed by extraction of nucleic acids from formalin-fixed paraffin-embedded blocks in NCKUH. Specimens were reviewed by pathologists who determined the percentage of viable tumor nuclei and adequacy for profiling mutation detection. Tumor deep targeted sequencing was performed by Oncomine Comprehensive Assays (OCA) (Thermo Fisher Scientific)^{19 (link)}. The OCA was designed to detect 143 solid tumor-related genes, including 73 hotspot genes, 49 focal CNV gains, 26 genes for full coding region sequencing (CDS), and 22 fusion driver genes. The Ion PGM Sequencing 200 Kit v.2 was used with the Ion PGM sequencer (Thermo Fisher Scientific) according to the manufacturer’s instructions. All samples were analyzed using the Torrent Suite Software 5.0.4, aligning all reads to the hg19 reference genome, and variant calling was performed running the Torrent Variant Caller plugin version 5.0.4.0.

DNA (300 ng for each sample) was fragmented using the Covaris S220 system (Covaris, Woburn, MA, USA) up to a final size of 300–500 bp according to the manufacturer’s recommendations. The DNA libraries of n01.08 and NG05 were prepared with the Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific, Waltham, MA, USA) for sequencing on the Ion Torrent PGM (Thermo Fisher Scientific, Waltham, MA, USA). The Ion PGM Template OT2 200 Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for emulsion PCR. DNA sequencing was performed using Ion 318 chip v2 and the Ion PGM Sequencing 200 Kit v2 (Thermo Fisher Scientific, Waltham, MA, USA).
Previously, WGS of N. gonorrhoeae clinical isolate i19.05 was done on a Roche Genome Sequencer GS FLX (Roche Holding, Basel, Switzerland) using a standard protocol for a shotgun genomic library. The accuracy of raw reads was improved with spectral alignment error correction tool SAET 3 and the Newbler v.2.6 (Roche Holding, Basel, Switzerland) was used for assembly.
The sequencing results were deposited in the NCBI database under PRJNA236643 and PRJNA243883 accession numbers. Accession numbers of assembled strains deposited to the NCBI Assembly database are GCA_000695425.1-i19.01, GCA_000705675.1-n01.08, and GCA_000763255.1-NG05.