Ion Torrent and Whole Exome Sequence

alexdem · 01-22-2014, 11:28 PM

Hi all,

I am a new user of an Ion Torrent and we are working on Whole Exome Sequencing (WES). From what I've read so far, GATK is the golden standard for such analysis when it comes to Illumina data BUT that does not apply so much to Ion Torrent data.
Does anyone have experience on WES with Ion Torrent and can help me by proposing software to use for my pipeline, or alterations on the GATK best practice?

Thank you in advance for your time

RemitoAmigo · 01-27-2014, 11:17 AM

Far from being a master in the field...Have you had a look at Ion Reporter?

alexdem · 01-27-2014, 11:25 AM

Well actually I have, but I am trying to find something open-source and not specifically from the producer (LifeTech).

NextGenSeq · 01-28-2014, 10:45 AM

You can generate FastQ files on the Ion Torrent. I would just use GATK.

The Broad has published ChIP-Seq on the PGM and they must have used GATK.

Zaag · 02-04-2014, 07:10 AM

GATK does not align reads so what do you use for this?

https://github.com/iontorrent/TS

Is open source TorrentSuite, contains a aligner and variant caller.

snetmcom · 02-04-2014, 10:17 AM

The Torrent Suite has specific settings for exome analysis already. I'm not sure why you would want to use other tools.

alexdem · 02-13-2014, 06:13 AM

Thank you for the suggestion. I will definitely try TorrentSuite!

Do you know if it the same software as the one running on Ion Proton?

GenoMax · 02-13-2014, 06:36 AM

AFAIK: Torrent suite download available on github does not include Ion reporter (which is not free software). If you have access to an ion machine then there is likely to be a copy there that you can use for the variant calling/coverage analysis.

alexdem · 02-13-2014, 06:41 AM

Thank you, I'll look into it.

BrianJames · 02-14-2014, 12:32 PM

Quote:

Originally Posted by alexdem

Thank you for the suggestion. I will definitely try TorrentSuite!

Do you know if it the same software as the one running on Ion Proton?

Yes, TS is the same as what is running your Ion Proton.
Brian

Jeremy · 02-17-2014, 09:24 PM

Ion reporter uses gatk doesn't it? Very little of the Ion torrent software is made by lifetech, most of it is just the freely available software with the default settings tweaked for torrent data.

BrianJames · 02-18-2014, 08:52 AM

I'm not sure what Ion Reporter uses, but the torrent server uses TMAP to align reads.

Jeremy · 02-18-2014, 07:06 PM

Quote:

Originally Posted by BrianJames

I'm not sure what Ion Reporter uses, but the torrent server uses TMAP to align reads.

Yeah, but TMAP is basically just a wrapper for BWA with the defualt being BWA fastmap.

snetmcom · 02-20-2014, 02:27 PM

TMAP is mostly BWA but it runs it in an iterative process. It also takes their flow bam files. I almost always see more data aligning with TMAP.

Ion Reporter and the server uses the same alignment and variant calling tools. The reporter software adds database annotation to the variants called with FreeBayes.

mbzmg1 · 04-16-2014, 04:45 PM

We have had success using GSNAP to map Ion Torrent reads -especially for RNAseq with splicing. Tophat2/Bowtie2 do not deal well with Ion Torrent data.

Brian Bushnell · 04-16-2014, 05:20 PM

I suggest you try BBMap - it can map RNA-seq data with very high error rates, including indels, and in my testing greatly outperforms GSNAP. For RNA-seq you have to include a few extra flags from default, as mentioned in that thread.

BBMap as compiled will handle reads up to 500bp. I don't really know how long Ion Torrent reads are so if that's too short, tell me and I can compile it with support for longer reads.

GenoMax · 04-16-2014, 05:28 PM

Quote:

Originally Posted by Brian Bushnell

BBMap as compiled will handle reads up to 500bp. I don't really know how long Ion Torrent reads are so if that's too short, tell me and I can compile it with support for longer reads.

Torrent reads are well below 500 bp (more like ~100- 150 bp) so this version should be ok.

mbzmg1 · 04-16-2014, 05:33 PM

Quote:

Originally Posted by Brian Bushnell

I suggest you try BBMap - it can map RNA-seq data with very high error rates, including indels, and in my testing greatly outperforms GSNAP. For RNA-seq you have to include a few extra flags from default, as mentioned in that thread.

BBMap as compiled will handle reads up to 500bp. I don't really know how long Ion Torrent reads are so if that's too short, tell me and I can compile it with support for longer reads.

Have you tested it against GSNAP with Ion Torrent data?
I will take a look and probably test it out if mismatch etc can be set as a percentage/fraction of read length. The problem with Tophat2/Bowtie2 and Ion Torrent data is a bias against longer reads which have more errors than shorter reads. This is also a slight problem with GSNAP as it will only allow a single indel. It would be great if BBMap allows for tweaking these parameters.

IonTom · 04-17-2014, 01:43 AM

Currently i am using NextGenMap for Alignment.
This works very well and has been adapted for varying read length and
the homopolymer error.

To make it behave closer to tmap you can try to increase --gap-read-penalty and --gap-ref-penalty.

I have not tried MOSAIK yet, which could also work well.

Brian Bushnell · 04-17-2014, 10:09 AM

Quote:

Originally Posted by mbzmg1

Have you tested it against GSNAP with Ion Torrent data?
I will take a look and probably test it out if mismatch etc can be set as a percentage/fraction of read length. The problem with Tophat2/Bowtie2 and Ion Torrent data is a bias against longer reads which have more errors than shorter reads. This is also a slight problem with GSNAP as it will only allow a single indel. It would be great if BBMap allows for tweaking these parameters.

I've tested it against GSNAP with synthetic Illumina data containing many indels and mismatches per read. BBMap has no limit to the number of indels or mismatches, but if there are too many the mapping score will drop below a cutoff and be rejected. A 150bp read can map with up to roughly 13 single-base-pair indels, or 29 snps, or a single deletion of up to (I think) 2.8 megabases, or some combination of things like that, while staying above the score cutoff. You do have to change the maxindel flag for RNA-seq, though; by default it is 16000; for vertebrates you should change it to around 200000 or so. Anyway, it can align reads just fine that spans 2 or 3 or even more introns.

BBMap is not biased against longer reads, because the scoring is always considered as a function of the read length; so a 100bp read with 1 mismatch would have the same score as a 200bp read with 2 mismatches. After factoring in ambiguity, the 200bp read might end up with a higher score, because a shorter read is more like to have multiple possible mapping locations, which incurs a score penalty.

Here's a comparison of BBMap and GSNAP:
https://drive.google.com/file/d/0B3l...it?usp=sharing

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01-22-2014, 11:28 PM	#1
alexdem Junior Member Location: Athens Join Date: Oct 2012 Posts: 4	Ion Torrent and Whole Exome Sequence Hi all, I am a new user of an Ion Torrent and we are working on Whole Exome Sequencing (WES). From what I've read so far, GATK is the golden standard for such analysis when it comes to Illumina data BUT that does not apply so much to Ion Torrent data. Does anyone have experience on WES with Ion Torrent and can help me by proposing software to use for my pipeline, or alterations on the GATK best practice? Thank you in advance for your time

04-17-2014, 01:43 AM	#19
IonTom Member Location: Germany Join Date: Apr 2014 Posts: 32	Currently i am using NextGenMap for Alignment. This works very well and has been adapted for varying read length and the homopolymer error. To make it behave closer to tmap you can try to increase --gap-read-penalty and --gap-ref-penalty. I have not tried MOSAIK yet, which could also work well. Last edited by IonTom; 04-24-2014 at 01:02 PM.

01-27-2014, 11:17 AM	#2
RemitoAmigo Member Location: Ottawa, Canada Join Date: Apr 2012 Posts: 22	Far from being a master in the field...Have you had a look at Ion Reporter?

01-27-2014, 11:25 AM	#3
alexdem Junior Member Location: Athens Join Date: Oct 2012 Posts: 4	Well actually I have, but I am trying to find something open-source and not specifically from the producer (LifeTech).

01-28-2014, 10:45 AM	#4
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	You can generate FastQ files on the Ion Torrent. I would just use GATK. The Broad has published ChIP-Seq on the PGM and they must have used GATK.

02-04-2014, 07:10 AM	#5
Zaag Senior Member Location: Amsterdam Join Date: Nov 2009 Posts: 112	GATK does not align reads so what do you use for this? https://github.com/iontorrent/TS Is open source TorrentSuite, contains a aligner and variant caller.

02-04-2014, 10:17 AM	#6
snetmcom Senior Member Location: USA Join Date: Oct 2008 Posts: 158	The Torrent Suite has specific settings for exome analysis already. I'm not sure why you would want to use other tools.

02-13-2014, 06:13 AM	#7
alexdem Junior Member Location: Athens Join Date: Oct 2012 Posts: 4	Thank you for the suggestion. I will definitely try TorrentSuite! Do you know if it the same software as the one running on Ion Proton?

02-13-2014, 06:36 AM	#8
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,091	AFAIK: Torrent suite download available on github does not include Ion reporter (which is not free software). If you have access to an ion machine then there is likely to be a copy there that you can use for the variant calling/coverage analysis.

02-13-2014, 06:41 AM	#9
alexdem Junior Member Location: Athens Join Date: Oct 2012 Posts: 4	Thank you, I'll look into it.

02-17-2014, 09:24 PM	#11
Jeremy Senior Member Location: Pathum Thani, Thailand Join Date: Nov 2009 Posts: 190	Ion reporter uses gatk doesn't it? Very little of the Ion torrent software is made by lifetech, most of it is just the freely available software with the default settings tweaked for torrent data.

02-18-2014, 08:52 AM	#12
BrianJames Member Location: San Diego Join Date: May 2010 Posts: 13	I'm not sure what Ion Reporter uses, but the torrent server uses TMAP to align reads.

02-20-2014, 02:27 PM	#14
snetmcom Senior Member Location: USA Join Date: Oct 2008 Posts: 158	TMAP is mostly BWA but it runs it in an iterative process. It also takes their flow bam files. I almost always see more data aligning with TMAP. Ion Reporter and the server uses the same alignment and variant calling tools. The reporter software adds database annotation to the variants called with FreeBayes.

04-16-2014, 04:45 PM	#15
mbzmg1 Junior Member Location: Colorado Join Date: Feb 2012 Posts: 9	We have had success using GSNAP to map Ion Torrent reads -especially for RNAseq with splicing. Tophat2/Bowtie2 do not deal well with Ion Torrent data.

04-16-2014, 05:20 PM	#16
Brian Bushnell Super Moderator Location: Walnut Creek, CA Join Date: Jan 2014 Posts: 2,707	I suggest you try BBMap - it can map RNA-seq data with very high error rates, including indels, and in my testing greatly outperforms GSNAP. For RNA-seq you have to include a few extra flags from default, as mentioned in that thread. BBMap as compiled will handle reads up to 500bp. I don't really know how long Ion Torrent reads are so if that's too short, tell me and I can compile it with support for longer reads.