Proton Sequencing

Ion Torrent Sequencing on S5

S5, an upgrade of Ion Torrent Proton, has been installed at the DNA Sequencing Facility in early 2017. It uses the same semiconductor technology with a simple sequencing chemistry (http://www.iontorrent.com). Both 200b and 400b sequencing are done on three different chips – 520, 530 and 540 with throughputs ranging from 5M – 100M reads and about 1Gb -12Gb. S5 is essentially an amalgamation of PGM and Proton; 318 chip (PGM) and PI chip (Proton) are equivalent to 520 and 540 chips respectively.

The main applications using different chips on S5 are similar to the ones on PGM with the difference that lot more multiplexing can be done on the former due to a 12-15 fold higher throughput. Human exome and whole RNA-seq can be done on 540 chip with multiplexing. All targeted gene panels can be put on S5 after pooling several more samples compared to the number pooled on 318 chip in PGM. Similarly, several large bacterial and viral genomes can be sequenced together on S5.

The major advantages of the technology are low cost and rapid turnaround. Only one sample is run at a time. So there is no waiting necessary to fill out a plate, a flow cell or a slide unlike other next generation sequencing platforms. The sequencing run times are only between 2.5 – 4 hours compared to few days run on Illumina.

Major Strength

Targeted sequencing of amplicons

• Uses AmpliSeq technology to design ultrahigh multiplex PCRs to sequence custom panel of genes
• Simple workflow, low cost – multiplex PCR based library preparation from FFPE samples
• Low DNA input – 10 ng DNA (minimum) unlike other platforms
• Quick turnaround – sequencing takes less than a day, only 1 sample/pool is run at a time
• Variant calling & annotation via point-N-click Torrent Suite & Ion Reporter software

Ion AmpliSeq Transcriptome Human Gene Expression

-includes more than 95% of human RefSeq genes with 10 ng RNA from FFPE and other samples

• A rapid, inexpensive gene expression analysis kit on FFPE and other samples
• 10 ng RNA input from fresh or frozen tissue
• Differential gene expression for more than 95% of human RefSeq genes
• Reproducible detection of high and low abundance transcripts from 20,802 genes
• Detection range exceeds microarray sensitivity
• Up to 8 multiplexed samples on a 540 chip (8 – 10M reads per sample)

Ion RNA-Seq for whole transcriptome or small RNA using same kit

-for identification of alternative splicing, transcript isoforms and fusion transcripts along with novel and low abundance transcripts with 100 ng – 1 µg total RNA

• Needs 100 ng – 1 µg total RNA
• Differential gene expression, discovery of novel & low abundance transcripts
• Identification of alternative splicing, transcript isoforms and fusion transcripts
• Up to 4 samples on a 540 chip (~20M reads per sample) depending on the depth of coverage required

Ion AmpliSeq Exome

• A simple and flexible workflow that targets ~33 Mb of coding exons
• Covers greater than 97% of coding regions as described by CCDS annotation
• Low DNA input of 50 ng per sample, up to 3 samples on a 540 chip with 75-100X mean coverage
• High percentage of on-target bases (>90%) with excellent uniformity

Ion TargetSeq Exome

• Hybridization based capture approach to make exome library
• Captures exons identified in several data bases e.g. Gencode, RefSeq, UCSC etc, functional RNA genes, predicted MiRNA binding sites and COSMIC annotated tumor variants
• A total of 46.2 Mb, up to 2 exomes can be multiplexed on a 540 chip

Comprehensive Cancer Panel

• Targets over 16,000 amplicons in 400 genes involved in tumor formation
• 95% of targets at 300X coverage using the 318 chip on PGM
• 6-8 samples pooled with 400-500X coverage on 540 chip

AmpliSeq Inherited Disease Panel

• Targets over 10,000 primer pairs to amplify the coding exons of 328 genes
• Over 700 unique inherited diseases included
• Major disease groups- neuromascular, cardiovascular, developmental and metabolic
• Multiple samples pooled on 540 chip depending on the coverage depth required.

AmpliSeq Custom Panel (www.ampliseq.com)

• Amplifies 24 to 3,072 amplicons in a single tube, target a few genes to hundreds of genes
• Ion AmpliSeq designer v5.6, is an online tool, to create and order custom panels
• The cumulative target sequence is up to 1 Mb.

Data Analysis

Torrent Suite, the Torrent server analysis pipeline is the primary software used to process raw data acquired by Proton to produce read files containing high quality bases. The base calls are in both SFF and FASTQ file formats for easy downstream analysis with third party analysis tools. The Torrent browser provides many matrices, graphs and reporting features derived from the pipeline results.

Filtering and trimming: This is done to remove low quality bases or uncertain base calls by filtering out entire reads and trimming low quality 3’ ends of reads respectively. The SFF files contain per-base quality scores along with all other read information. The FASTQ files also provide per-base quality scores.

TMAP: Torrent Mapping Alignment Program implements a two-stage mapping approach – reads that do not align during the 1st stage are passed to the 2nd stage with a new set of algorithms and/or parameters. Overall the alignment provides an index to determine run and library quality.

Variant Caller: After analysis the variant caller generates a report of the SNVs and the insertion-deletions obtained from the data set (Torrent Suite User Documentation, Life Technologies).

Ion Reporter: Provides annotated variant calls with various filtering options by using different databases.

Commercial software NextGene, DNASTAR, PARTEK and other open source software are available for analysis of Proton data.

We provide data analysis service for a fee using Torrent suite, Ion Reporter, NextGene, and a number of open source software.

§ The sequencing charges include emPCR, sequencing, and preliminary data analysis to provide FASTQ, BAM and BAI files, as well as sequence alignment and variant calls.

Library prep & QC not included.

Note – Downstream data analysis (provided after consultation with the user): $100/hr

Please contact Tapan Ganguly at 215-573-7238, or gangulyt@pennmedicine.upenn.edu for consultation.

Penn Genomics Analysis Core is an Abramson Cancer Center Shared Resource that is approved and partially funded by the National Cancer Institute.