Effects of the Ion PGM™ Hi-Q™ sequencing chemistry on sequence data quality

Abstract

Massively parallel sequencing (MPS) offers substantial improvements over current forensic DNA typing methodologies such as increased resolution, scalability, and throughput. The Ion PGM™ is a promising MPS platform for analysis of forensic biological evidence. The system employs a sequencing-by-synthesis chemistry on a semiconductor chip that measures a pH change due to the release of hydrogen ions as nucleotides are incorporated into the growing DNA strands. However, implementation of MPS into forensic laboratories requires a robust chemistry. Ion Torrent's Hi-Q™ Sequencing Chemistry was evaluated to determine if it could improve on the quality of the generated sequence data in association with selected genetic marker targets. The whole mitochondrial genome and the HID-Ion STR 10-plex panel were sequenced on the Ion PGM™ system with the Ion PGM™ Sequencing 400 Kit and the Ion PGM™ Hi-Q™ Sequencing Kit. Concordance, coverage, strand balance, noise, and deletion ratios were assessed in evaluating the performance of the Ion PGM™ Hi-Q™ Sequencing Kit. The results indicate that reliable, accurate data are generated and that sequencing through homopolymeric regions can be improved with the use of Ion Torrent's Hi-Q™ Sequencing Chemistry. Overall, the quality of the generated sequencing data supports the potential for use of the Ion PGM™ in forensic genetic laboratories.

Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias

“…Salipante et al sequenced the V1-2 region of the 16S rRNA gene in the equimolar 20-species mock community and found a higher error rate (especially in homopolymeric regions) from the Ion Torrent PGM sequencing data set compared with the corresponding Illumina MiSeq sequencing data set (Salipante et al, 2014). It is important to note that since the publication by Salipante et al, the Ion Torrent Hi-Q chemistry has been introduced, which significantly improves the sequence read quality and error rates in homopolymeric regions (Churchill et al, 2016; Pereira et al, 2016). In the study by Salipante et al, the authors also noted the deviation in relative abundance from the expected 5% for some of the included species.…”

Microbial Assemblages in Pressurized Antarctic Brine Pockets (Tarn Flat, Northern Victoria Land): A Hotspot of Biodiversity and Activity

“…PCR products were purified, quantified, pooled for emulsion PCR and sequenced as described in Papale et al [44]. The Ion Torrent PGM platform (Life Technologies, USA) with Hi-Q sequencing chemistry was used to reduce the problem of poor homopolymer sequencing [48,49]. All steps during amplification and sequencing were checked using negative controls.…”

Parsing apart the contributors of mitochondrial DNA mixtures with massively parallel sequencing data

“…The http://dx.doi.org/10.1016/j.fsigss.2017.09.145 Received 7 September 2017; Accepted 19 September 2017concordance data were generated via long-PCR on the Ion PGM and MiSeq (Illumina, San Diego, CA, USA) sequencers as described previously in Churchill et al[7] and King et al[1].…”