Best RNA-seq program for unpaired reads

reshetovdenis · 06-04-2010, 04:23 AM

Hi everyone,
I have RNA-seq unpaired reads from Solid machine. The read length is 35.
Please, help me: I can't find any software except ABI (that works on PBS and LSF clusters) to analyse the data. I do have access to a cluster machine but it has no PBS or LSF.

kopi-o · 06-04-2010, 09:49 AM

There are many aligners that can deal with SOLiD's color space format nowadays. Google for BFAST, BWA, Bowtie or Mosaik, for example.

reshetovdenis · 06-04-2010, 09:54 AM

kopi-o, thanks for reply. I've seen these programs and used Bowtie. But they are good for sequencing genomes not for RNA-seq data, am I wrong?

Richard Finney · 06-04-2010, 09:59 AM

If the data is small enough, use blat. Access to a biowulf cluster can help if it's big.
This will take a long time.
You can prefilter using a SR aligner (e.g.: bwa).
Map to genome. Map against refseq. Merge outputs. (thest steps are hard to do).
Then blat the ones that don't map ... and merge again.
Not easy, but doable.

mrawlins · 06-04-2010, 12:07 PM

What we've used on our SOLiD data (50bp reads) is the BioScope WT analysis pipeline. I am impressed with BowTie from a technical perspective, and it seems to give good alignment, but it isn't able to do junction mapping (i.e., you'll get no splicing information, and all reads that cover a splice junction will go unmapped, or be mapped erroneously).
I haven't seen any software able to do the junction mapping in colorspace other than BioScope or the Corona Lite WT pipeline, from ABI. Some people are comfortable using the "double-encoding" trick and using any old junction mapper, but I remain unconvinced of the accuracy and reliability of such a method. If I didn't need splicing information, I would go with BowTie. Since I sometimes do, I'm sticking with ABI's software.

In my estimation it's worth getting TORQUE/maui up and running on your cluster and using ABI's software.

And if you find something else that will do junction mapping on SOLiD reads, I'd love to hear about it.

kopi-o · 06-04-2010, 01:53 PM

As far as I know, though. Bioscope uses a splice junction database to align against, rather than finding splice junctions de novo, so it is not really conceptually different from using another aligner and adding artificially constructed exon-exon junctions to your reference genome (although that takes some extra effort, of course). The previous versions of the ABI pipeline, though, chopped up the reads into two pieces and mapped them separately, which enabled de novo splice junction detection.

jwfoley · 06-06-2010, 08:08 PM

Quote:

Originally Posted by reshetovdenis

kopi-o, thanks for reply. I've seen these programs and used Bowtie. But they are good for sequencing genomes not for RNA-seq data, am I wrong?

Other way around. These are short-read aligners that simply map each tag against the reference sequence; they're designed for quantitative, targeted genomics like RNA-seq or ChIP-seq, but basically useless for de novo assembly.

Bowtie and BFAST can both handle SOLiD data. BWA's treatment of colorspace is an ugly hack and I wouldn't use it.

Pejman · 10-01-2010, 05:42 AM

Quote:

Originally Posted by mrawlins

And if you find something else that will do junction mapping on SOLiD reads, I'd love to hear about it.

TopHat-ers say that TopHat's is gonna be able to handle colorspace data very soon.

zee · 10-01-2010, 06:51 AM

Try NovoalignCS (www.novocraft.com) it's quite sensitive and has many useful features for SOLiD reads. There's also documentation on how to do alignment to a full genome.
NovoalignCS will do mismatches and indels with colorspace reads and supports csfasta/qual formats.

Pejman · 10-01-2010, 07:36 AM

Seems nice but I guess NovoalignCS needs a license.

zee · 10-01-2010, 08:50 AM

Yes it does but it's free and IMO worth a try if you're evaluating accurate tools for your work.

Cole Trapnell · 10-04-2010, 09:20 AM

Quote:

Originally Posted by Pejman

TopHat-ers say that TopHat's is gonna be able to handle colorspace data very soon.

We released a version of TopHat that supports Colorspace yesterday (10/3)

Pejman · 10-04-2010, 09:37 AM

Quote:

Originally Posted by Cole Trapnell

We released a version of TopHat that supports Colorspace yesterday (10/3)

wow, cool I knew that it's gonna be released soon but, cool! We are not interested in new splice junctions, but Bowtie output does not fit to Cufflinks, so why not TopHat.

I'm now trying out NovoalignCS, do you have any comparisons with other pipelines already? Let's say assuming that we don't care about novel splice junctions, just simple RNAseq for differential expression analysis.

jgibbons1 · 10-04-2010, 12:39 PM

I really like the program rSeq by Hui Jiang.

1st convert your reads to standard fasta format. The software has a mapping program and then a script that calculates RPKM for each reference transcript.

Also...its free!

danielr · 10-06-2010, 09:07 AM

Quote:

Originally Posted by Pejman

wow, cool I knew that it's gonna be released soon but, cool! We are not interested in new splice junctions, but Bowtie output does not fit to Cufflinks, so why not TopHat.

You get the right output format with the -S option in Bowtie (TopHat has nice splicing-related features Bowtie lacks though).

Aman Mahajan · 02-02-2012, 01:18 AM

Quote:

Originally Posted by jgibbons1

I really like the program rSeq by Hui Jiang.

1st convert your reads to standard fasta format. The software has a mapping program and then a script that calculates RPKM for each reference transcript.

Also...its free!

I am having problem running RSEQtools. This is the installation procedure.

Installation
============

The installation steps have to be executed in the following order:

1) Install the GNU Scientific Library

( I have done the 1st step)

2) Install BIOS - Have not been able to carry out this step.

- Set the following environment variables:
BIOINFOCONFDIR=/pathTo/RSEQtools/bios/conf
BIOINFOGSLDIR=/pathTo/gsl-X.XX

- Compiling BIOS:
cd pathTo/RSEQtools/bios
make
make prod

Thanks .

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06-04-2010, 04:23 AM	#1
reshetovdenis Junior Member Location: Moscow Join Date: May 2010 Posts: 2	Best RNA-seq program for unpaired reads Hi everyone, I have RNA-seq unpaired reads from Solid machine. The read length is 35. Please, help me: I can't find any software except ABI (that works on PBS and LSF clusters) to analyse the data. I do have access to a cluster machine but it has no PBS or LSF.

06-04-2010, 09:49 AM	#2
kopi-o Senior Member Location: Stockholm, Sweden Join Date: Feb 2008 Posts: 319	There are many aligners that can deal with SOLiD's color space format nowadays. Google for BFAST, BWA, Bowtie or Mosaik, for example.

06-04-2010, 09:54 AM	#3
reshetovdenis Junior Member Location: Moscow Join Date: May 2010 Posts: 2	kopi-o, thanks for reply. I've seen these programs and used Bowtie. But they are good for sequencing genomes not for RNA-seq data, am I wrong?

06-04-2010, 09:59 AM	#4
Richard Finney Senior Member Location: bethesda Join Date: Feb 2009 Posts: 700	If the data is small enough, use blat. Access to a biowulf cluster can help if it's big. This will take a long time. You can prefilter using a SR aligner (e.g.: bwa). Map to genome. Map against refseq. Merge outputs. (thest steps are hard to do). Then blat the ones that don't map ... and merge again. Not easy, but doable.

06-04-2010, 12:07 PM	#5
mrawlins Member Location: Retirement - Not working with bioinformatics anymore. Join Date: Apr 2010 Posts: 63	What we've used on our SOLiD data (50bp reads) is the BioScope WT analysis pipeline. I am impressed with BowTie from a technical perspective, and it seems to give good alignment, but it isn't able to do junction mapping (i.e., you'll get no splicing information, and all reads that cover a splice junction will go unmapped, or be mapped erroneously). I haven't seen any software able to do the junction mapping in colorspace other than BioScope or the Corona Lite WT pipeline, from ABI. Some people are comfortable using the "double-encoding" trick and using any old junction mapper, but I remain unconvinced of the accuracy and reliability of such a method. If I didn't need splicing information, I would go with BowTie. Since I sometimes do, I'm sticking with ABI's software. In my estimation it's worth getting TORQUE/maui up and running on your cluster and using ABI's software. And if you find something else that will do junction mapping on SOLiD reads, I'd love to hear about it.

06-04-2010, 01:53 PM	#6
kopi-o Senior Member Location: Stockholm, Sweden Join Date: Feb 2008 Posts: 319	As far as I know, though. Bioscope uses a splice junction database to align against, rather than finding splice junctions de novo, so it is not really conceptually different from using another aligner and adding artificially constructed exon-exon junctions to your reference genome (although that takes some extra effort, of course). The previous versions of the ABI pipeline, though, chopped up the reads into two pieces and mapped them separately, which enabled de novo splice junction detection.

10-01-2010, 06:51 AM	#9
zee NGS specialist Location: Malaysia Join Date: Apr 2008 Posts: 249	Try NovoalignCS (www.novocraft.com) it's quite sensitive and has many useful features for SOLiD reads. There's also documentation on how to do alignment to a full genome. NovoalignCS will do mismatches and indels with colorspace reads and supports csfasta/qual formats.

10-01-2010, 07:36 AM	#10
Pejman Member Location: Switzerland Join Date: Jul 2010 Posts: 23	Seems nice but I guess NovoalignCS needs a license.

10-01-2010, 08:50 AM	#11
zee NGS specialist Location: Malaysia Join Date: Apr 2008 Posts: 249	Yes it does but it's free and IMO worth a try if you're evaluating accurate tools for your work.

10-04-2010, 12:39 PM	#14
jgibbons1 Senior Member Location: Worcester, MA Join Date: Oct 2009 Posts: 133	I really like the program rSeq by Hui Jiang. 1st convert your reads to standard fasta format. The software has a mapping program and then a script that calculates RPKM for each reference transcript. Also...its free!