Tophat paired end alignment.

[adeslat]

Dear forum,

I have RNASeq SOLID data that is paired end. I want to use Tophat to perform the alignment. Does anyone out there know how to
ensure that "even if an end of a pair could not be aligned, it should still have an entry"

Aaron Quinlan requested that I ensure that this is so -- because I went to use BEDtools with the -pe option and it failed saying my data was not sorted -- he suggested that it may be because I did not ensure that the pair every seq had an entry even if it was not aligned....

Best,
Anne

[Jon_Keats]

Check the sam header SO identifier. In the current release the sort nomenclature is not matching the SAM specification and will cause some applications the through errors. Either resort using picard, not samtools, or just use sed to modify the SO header to conform to specification

[adeslat]

Check the sam header SO identifier. In the current release the sort nomenclature is not matching the SAM specification and will cause some applications the through errors. Either resort using picard, not samtools, or just use sed to modify the SO header to conform to specification

Jon,
Of all the people to reply -- thanks -- I've been reading your various how-tos -- they are simply wonderful. Thank you -- I will do as you suggest and report back -- picard works better?

Best,
Anne

[Jon_Keats]

In the case of this error that I discovered trying to get picard to remove the duplicates I found that samtools did not correct the SO header while picard did, not sure why that occurs as a bwa generated bam file when sorted with samtools gets the right header.

[adeslat]

elsewhere documented errors with samtools

Hi Jon,

I was looking at "Scripture" and found looking through their course material at:

http://molecularevolution.org/resources/activities/scripture_activity

the following:

"There is currently an incompatibility between samtools sort and Picard (which is used by Scripture); therefore you need to sort the BAM file using Picard and not samtools:"

By the way -- the instructions on this site are spot on, clean and complete.

The reference is the same Nature Biotechnology issue where the Steven Salzberg and Cole Trapnell so nicely introduced the "Tuxedo" suite in the letter "Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation"
Cole Trapnell, Brian A Williams,Geo Pertea, Ali Mortazavi, Gordon Kwan, Marijke J van Baren, Steven L Salzberg, Barbara J Wold & Lior Pachter
Nature Biotechnology 28, 511–515 (2010) doi:10.1038/nbt.1621

Scripture is discussed in:
"Ab initio reconstruction of cell type–specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs" - pp503 - 510
Mitchell Guttman, Manuel Garber, Joshua Z Levin, Julie Donaghey, James Robinson, Xian Adiconis, Lin Fan, Magdalena J Koziol, Andreas Gnirke, Chad Nusbaum, John L Rinn, Eric S Lander & Aviv Regev
doi:10.1038/nbt.1633

Both are excellent articles....

Anne

[Joker!sAce]

Hi,

I looked at this tutorial and was looking for the "scripture_alpha2.jar" file. I cannot find that file anywhere. Any insights or help would be appreciated.

Thanks!