RNA-Seq: TopHat with Roche/454 reads

[DNAjunk]

Hellow

I have got Roche/454 RNA-Seq reads from human samples.
It seems that TopHat/Cufflinks is one of the most used pipeline for analyzing RNA-Seq data, however, for short reads only (non-454 reads).
The program Bowtie, which is called by TopHat, cannot map 454 reads.

Still I would like to take adavantage of TopHat's features if possible.
Has anybody tried to use 454 reads with TopHat, implemented a workaround, or are there any alternatives to TopHat/Bowtie that yield good results for 454 reads?

Any help, comments are appreciated!

[kmcarr]

Here is an item from the release notes for TopHat v 1.2.0, just released on 1/18/2011

Version 1.2.0 also addresses the following issues:

* A problem with alignment of reads of mixed length has been fixed. Users with a wide range of read lengths, such as trimmed 454 data sets, are strongly encouraged to upgrade.

So it would appear the 454 reads can be used with TopHat.

[DNAjunk]

I have used the latest version. But Bowtie is still not able to map to long 454 reads in the first mapping round. Surely, for the IUM (initially unmapped reads) which comprise the majority of the 454 reads, TopHat chops them into smaller parts that can be mapped by bowtied. But I think that it would be better if one could use the full length 454 reads.

[Jeremy]

Is there any reason you don't want to use the 454 software? Newbler is very good.

[DNAjunk]

Can newbler detect new splice variants ?

[Jeremy]

A cDNA de-novo assembly gives an output of isotigs (essentially mRNAs) grouped into isogroups (representing genes) by traversing the contig graph (each contig theoretically represents an exon) which gives you a list of the contigs/exons per isogroup/gene and the combinations of them that form the isotigs/mRNAs. Of course 454 errors make this all much less straightforward than it sounds.

Not sure if there is an easy way to get to new splice variants from here, but I do know that gsMapper allows you to use a fully annotated genome as a reference including gene names, exon positions and known SNPs which may allow for contigs to be mapped to exons.

[sulicon]

I have met the same problem. I have also had a look at G-Mo.R-Se, a Tophat alternative. Again, the aligner it uses -- SOAP, only works with short reads...

Still looking for a splicing junction discovery tool which is independent of the aligners, i.e. works with SAM/BAM files directly.

[sulicon]

It seems that "MapSplice" fits my needs very well. It splits the raw reads into fragments first, so it could deal with both short and long reads.

http://www.netlab.uky.edu/p/bioinfo/MapSplice

[Jeremy]

The long reads of the 454 are very informative for exon junction sites. I would try to find a method that makes use of this information rather than discarding it by splitting up the reads.

[greenshell]

The long reads of the 454 are very informative for exon junction sites. I would try to find a method that makes use of this information rather than discarding it by splitting up the reads.

MapSplice splits the raw reads into fragments first, but the fragments are not processed independently. After each fragment is aligned, the alignments of all fragments of a read will be assembled to form alignments as long as possible

[prisnirath]

Help with Roche454 data and Bowtie!

I am working with Roche 454data and am trying to align sequences using Bowtie.

$ bowtie -k 2 -a BRCA1_coding -q 1FR.fastq

But I am getting No alignments.

# reads processed: 1911
# reads with at least one reported alignment: 0 (0.00%)
# reads that failed to align: 1911 (100.00%)
No alignments

Is there any way to obtain atleast a few alignments?
Or does Bowtie not support 454 data alignment??

[jochensupper]

Our mapper (Genomatix) has a local spliced alignment mode that allows to align complete transcripts to the genome without prior split of the reads (it works fine for 454 reads). Attached is a screenshot of assembled (velvet) RNA-Seq reads mapped to a reference genome.

By the way there is another thread discussing a similar topic:

http://seqanswers.com/forums/showthread.php?t=11458

[sahusarika]

Roche454

Dear all
i an beginner in NGS pls help me.
i hv roche 454 data in 10 set in all different condition and i hv to do trancriptome analysis . i m clc genomic software so pls help me how to proceed the task