Ion Torrent PGM Sequencing

An Ion Torrent sequencer (PGM – Personal Genome Machine) has been installed at the DNA Sequencing Facility, School of Medicine toward the end of 2011. Ion Torrent combines pre-existing semiconductor technology with a simple sequencing chemistry ( http://www.iontorrent.com ). The sequencer is essentially a solid state pH meter. Known for scalability, simplicity and speed, this inexpensive technology provides an array of options from 500K reads on a 314 chip to 5M reads on

a 318 chip as well as a read length of 200 and 400b. The sequencing runs are only 4 – 8 hours compared to few days run on Illumina. 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 new generation sequencing platforms.

Major Strength

Targeted sequencing of amplicons

• Use 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
• Several off-the shelf targeted gene panels available

Other Applications

Resequencing of barcoded multiplexed samples,

• Sequencing of captured library (captured region 100 Kb to 1 Mb) using 316 and 318 chips
• Sequencing of bacterial and viral genomes
• Sequencing of metagenomic samples with 200 and 400b sequencing chemistry
• RNA-seq specially small RNA sequencing
• Validation of sequence data obtained on other platforms
• Low cost sequencing of BAC clones and other constructs e.g. gene targeting vector

AmpliSeq Cancer Hotspot Panel v2

Ion Torrent offers an AmpliSeq Cancer panel of 50 most common cancer genes with 2,800 COSMIC mutations. 207 amplicons can be amplified in a single tube using 10 ng of DNA including DNA from FFPE tissues. The subsequent libraries after barcoding and pooling can be run on a 314, 316 or 318 chip after emulsion PCR of the library fragments.

Comprehensive Cancer Panel

This expanded panel targets over 400 genes involved in tumor formation with 95% of targets at 300X coverage using the 318 chip.

AmpliSeq Inherited Disease Panel

This employs more than 10,000 primer pairs to amplify the coding exons of 328 genes associated with over 700 unique inherited diseases including neuromascular, cardiovascular, developmental and metabolic diseases. The panel can be run on 316 or 316 chip with 200 b sequencing chemistry.

AmpliSeq Custom Panel (www.ampliseq.com)

This will amplify 24 to 3,072 amplicons in a single tube, perfect for targeting a few genes to hundreds of genes. The Ion AmpliSeq designer v1.2, an online tool, allows users to create and order custom panels designed with their genes of interest. The cumulative target sequence can be up to 1 Mb and the input DNA is just 10 ng.

Runs Performed

We have done large number of sequencing projects with captured DNA, small RNA libraries and cancer panel genes using DNA from FFPE tissues as well as custom panel of genes. The results have been quite satisfactory in terms of throughput and read lengths. The sequencing runs performed at the core have led to a number of publications.

Data Analysis

Torrent Suite, the Torrent server analysis pipeline is the primary software used to process raw data acquired by PGM sequencer 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 others are available for analysis of PGM data.

We provide data analysis service for a fee using Torrent suite, NextGene and other software.

§ The sequencing price includes 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

Ion Torrent PGM Sequencing Request Form (word doc.)

Please contact Tapan Ganguly at 215-573-7238, e-mail: gangulyt@mail.med.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.