Ion Torrent Sequencing

An Ion Torrent sequencer (Personal Genome Machine – PGM) has been installed at the DNA Sequencing Facility, School of Medicine recently. 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 is really moving fast to achieve new goals in terms of throughput and read length. 

The main applications of this technology at this point include

  • Resequencing of barcoded samples,
  • Sequencing of captured library (captured region 100 Kb to 1 Mb) using 316 chip
  • Sequencing of bacterial and viral genomes
  • Sequencing of metagenomic samples
  • RNA-seq specially small RNA sequencing
  • Validation of sequence data obtained on other platforms

AmpliSeq Cancer panel
Ion Torrent has introduced an AmpliSeq Cancer panel of 46 most common cancer genes with 739 known mutations. 190 amplicons can be amplified in a single tube using 10 ng of DNA including DNA from FFPE tissues. The subsequent library can be run on a 314 or 316 chip after emulsion PCR of the library fragments.

In Development
An expanded panel will target over 400 genes involved in tumor formation with 95% of targets at 100X coverage using the 318 chip.

AmpliSeq Custom Panel

In Development
This will amplify up to 1536 targets in a single tube.

AmpliSeq Inherited Disease Panel

In Development
This will target genes implicated in over 140 inherited diseases at approximately 30X coverage using the 316 chip.

Runs Performed So Far
So far we have done a number of sequencing runs with captured DNA and small RNA libraries using 100 b (read length) sequencing kit. The results have been quite encouraging in terms of throughput. One run of a captured library on 314 chip produced 5-fold more bases and 4-fold more reads compared to the chip’s specifications. The runs on 316 chips also far outperformed the cited specifications. The 318 chip with 1 Gb output has already been released. The 200 b read length sequencing option would be available soon.

We are ready to accept samples now for sequencing on Ion Torrent. 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. There should be a rapid turn-around as the sequencing runs are only 2 hours.

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.

AmpliSeq Cancer 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).

Commercial software NextGene, DNASTAR, PARTEK and others are available for analysis of PGM data. 

Expected Run Results (100 b read length)

Sequencing Substrate

314 chip

316 chip

318 chip

Throughput

> 10 Mb

> 100 Mb

> 500 Mb

No of Reads

> 100,000

> 1 M

> 5 M

Introductory Price Effective Jan 1, 2012

Sequencing Chip                                                314                 316                 318

* gDNA (including long amplicons, > 400 b)   $500              $700               $950

§  Amplicons (< 250 b)                                                $400              $600               $850

* AmpliSeq Cancer panel                                $600

(46 cancer genes, 739 known mutations)                                                      

* The price includes library prep (for gDNA and amplicons), quality assessment, emPCR, and sequencing on a chip.

§  The price does not include the preparation of amplicon library with Ion adaptors.

Note
1. Pricing for 200 b sequencing runs will be available soon.
2. Data analysis can be provided after consultation with the user at an additional cost of $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 to set up a meeting.