SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Pacific Biosciences



Similar Threads
Thread Thread Starter Forum Replies Last Post
BBMap (aligner for DNA/RNAseq) is now open-source and available for download. Brian Bushnell Bioinformatics 665 Today 03:50 AM
PacBio Library Prep workshop and PacBio SMART-Portal bootcamp - UC Davis - April 2015 DNATECH Events / Conferences 1 04-02-2015 08:33 AM
RNAseq by PacBio Ddhamburger RNA Sequencing 2 06-29-2012 03:17 AM
who coined RNAseq? RNAseq as an alignment first approach only brachysclereid Bioinformatics 3 01-10-2012 12:17 PM

Reply
 
Thread Tools
Old 11-20-2018, 09:27 AM   #1
RamP
Junior Member
 
Location: Hyderabad

Join Date: Feb 2013
Posts: 3
Default Aligner for PacBio RNASeq

I am wondering if I can use BLASR for aligning RNASeq reads from Human cell lines. I am interested in identifying the splice sites. I read this paper "Evaluation of tools for long read RNA-seq splice-aware alignment" (Bioinformatics, 2018) where different splice aware tools were compared and concluded GMAP to be the best among the compared ones. However, they didn't include BLASR in their evaluation. Could the reason be that BLASR is suitable only for genomic read alignments? Thanks.
RamP is offline   Reply With Quote
Old 02-04-2019, 01:33 PM   #2
Magdoll
Member
 
Location: Bay Area

Join Date: Aug 2011
Posts: 30
Default

You cannot use BLASR. You can use minimap2, GMAP, STAR, etc.
A tutorial for these aligners for PacBio RNA (Iso-Seq) data is available here:
https://github.com/Magdoll/cDNA_Cupc...AP,-STAR,-BLAT
Magdoll is offline   Reply With Quote
Old 02-04-2019, 06:08 PM   #3
RamP
Junior Member
 
Location: Hyderabad

Join Date: Feb 2013
Posts: 3
Default

Quote:
Originally Posted by Magdoll View Post
You cannot use BLASR.
Thanks for your response and the link. What could be the reason for BLASR being not a suitable aligner? I mean, it works like BLAST finding local alignments after incorporating the PacBio error model and that should be good enough to align exon regions on the genome. What am I missing here? Thanks.
RamP is offline   Reply With Quote
Old 02-04-2019, 07:27 PM   #4
Magdoll
Member
 
Location: Bay Area

Join Date: Aug 2011
Posts: 30
Default

BLASR is not splice aware - it cannot map exon-exons. Newer aligners like minimap2 are well equipped to handle errors for PacBio data. For most PacBio RNA-seq (what we call Iso-Seq) data, the error rate would be very low anyway.

In more practical terms, BLASR is getting phased out. We encourage users to switch to newer aligners like minimap2 that are being actively maintained and developed.
Magdoll is offline   Reply With Quote
Old 02-04-2019, 11:56 PM   #5
RamP
Junior Member
 
Location: Hyderabad

Join Date: Feb 2013
Posts: 3
Thumbs up

Thanks for taking time explaining this. One last thing.
Would Minimap2 be effective enough aligning to non-canonical coding regions, for example, fusion transcripts?
Perhaps, I should give it a try myself, but thought I would ask anyway.
Thanks again.

Last edited by RamP; 02-05-2019 at 12:03 AM.
RamP is offline   Reply With Quote
Old 02-05-2019, 12:35 PM   #6
Magdoll
Member
 
Location: Bay Area

Join Date: Aug 2011
Posts: 30
Default

Yes both GMAP and minimap2 could work for fusion detection.

In GMAP it would be using the `-n 0` parameter which says "only output a single alignment *unless* the best alignment is chimeric" which essentially screens for fusion transcripts when you look for sequences with 2+ alignment results.

In minimap2 I think it's `--secondary=no`, though admittedly I have tested it less.

I have a follow up script that processes the SAM files to look for fusion: https://github.com/Magdoll/cDNA_Cupc...ng-step#fusion

If you have problems using the scripts above, please file through GitHub Issues.
Magdoll is offline   Reply With Quote
Reply

Tags
blasr, pacbio, rna-seq

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 09:25 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO