University of Pennsylvania
DNA Sequencing Facility
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 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 multiplexed 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 with 200 b 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 Panel v2
Ion Torrent has introduced 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 or 316 chip after emulsion PCR of the library fragments.
Comprehensive Cancer Panel now available
This expanded panel targets over 400 genes involved in tumor formation with 95% of targets at 300X coverage using the 318 chip.
AmpliSeq Custom Panel
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.
AmpliSeq Inherited Disease Panel just released
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 318 chip with 200 b sequencing chemistry.
Runs Performed So Far
So far we have done a number of sequencing projects with captured DNA, small RNA libraries and cancer panel genes using DNA from FFPE tissues. 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 throughputs on 318 chips are also higher than the specification. Currently we are offering 200 b sequencing option.
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 new generation sequencing platforms. The sequencing runs are only 2 hours compared to overnight run on 454 and few days run on Illumina or SOLID.
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.
We provide data analysis service for a fee using Torrent suite, NextGene and other software.
Expected Run Results (400 b sequencing only on 314 chip currently)314 Chip |
316 Chip |
318 Chip |
||
|---|---|---|---|---|
| Read Length | 200b |
400b |
200b |
200b |
| No. of Reads* | > 100,000 |
> 100,000 |
> 1M |
~ 5 M |
| Throughput* | >20 Mb |
> 40 Mb |
>200 Mb |
~ 1 Gb |
Pricing Effective April 1, 2013
| Sequencing Chip | 314 |
316 |
318 |
|---|---|---|---|
| *gDNA (& also amplicons, includes 1 library prep) | $600 (200b) $725 (400b) |
$800 (200b) |
$995 (200b) |
| § Amplicons (library with adaptors already made) | $515 (200b) $640 (400b) |
$720 (200b) |
$920 (200b) |
| *AmpliSeq Cancer panel v2 ( 50 cancer genes, 2,800 COSMIC mutations, includes 1 library prep) | $700 (200b) |
$900 (200b) |
|
| Contact us for multiplexing | |||
| AmpliSeq Comprehensive Cancer & other panels | Contact us | ||
| Barcoded library - $80 (gDNA, long amplicons, 4 minimum) | $170 (for AmpliSeq cancer or custom panel, 4 minimum) | ||
§The price does not include the preparation of amplicon library with Ion adaptors but includes sample evaluation.
Note - Data analysis can be provided after consultation with the user at an additional cost of $100/hr.
Please contact Tapan Ganguly at 215-573-7238, email: gangulyt@mail.med.upenn.edu to set up a meeting.
Ion Torrent PGM Sequencing Request Form
The DNA Sequencing Facility is an Abramson Cancer Center Shared Resource that is approved and partially funded by the National Cancer Institute.
