Question: Tophat Rna-Seq Mapping Quality
1
gravatar for siyu
5.7 years ago by
siyu130
China
siyu130 wrote:

if a read can be mapped to more than one location in reference genome,so-called mulit-read , what's the read's mapping quality reported by TopHat ??

how about BWA?

thanks so much?

tophat mapping rna-seq quality • 7.4k views
ADD COMMENTlink modified 5.0 years ago by zju.whw30 • written 5.7 years ago by siyu130
1
gravatar for predeus
5.4 years ago by
predeus770
Russia
predeus770 wrote:

This actually does not seem to hold upon checking (or at least not anymore). In Tophat 1.4.1, there is no mapping quality of 2. I've looked at a large number of BAM files and it seems that the qualities are as follows:

  • 255 = unique mapping
  • 3 = maps to 2 locations in the target
  • 1 = maps to 3-4 locations
  • 0 = maps to 5 or more locations (up to the number defined in "--prefilter-multihits").

cheers

ADD COMMENTlink modified 5.4 years ago • written 5.4 years ago by predeus770

Interesting, thanks for doing the legwork!

ADD REPLYlink written 5.4 years ago by Mikael Huss4.6k
0
gravatar for Mikael Huss
5.7 years ago by
Mikael Huss4.6k
Stockholm
Mikael Huss4.6k wrote:

For Tophat:

From http://seqanswers.com/forums/showthread.php?t=10624:

255 = unique mapping

3 = maps to 2 locations in the target

2 = maps to 3 locations

1 = maps to 4-9 locations

0 = maps to 10 or more locations.

I think this is correct, it fits with what I have observed.

ADD COMMENTlink written 5.7 years ago by Mikael Huss4.6k

Thank you so much !

ADD REPLYlink written 5.7 years ago by siyu130
0
gravatar for zju.whw
5.0 years ago by
zju.whw30
China
zju.whw30 wrote:

My tophat output bam file is using 50 to indicate the unique mapping, is there any official matrials about the mapping quality of unique mapped reads?

In tophat website http://tophat.cbcb.umd.edu/, it says that "most of the optional SAM fields (AS, MD, NM, and etc.) generated by Bowtie 2 are now reported by TopHat as well (reconstructed as necessary)".

And in bowtie2 website http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#mapping-quality-higher-more-unique, it says that "Mapping quality: higher = more unique".

However, I cannot find the exact mapping quality number for unique mapped reads

ADD COMMENTlink written 5.0 years ago by zju.whw30

As mentioned above the unique mapping should be scored 255. I don't know why your BAM file has score of 50 for unique alignments. Did you generate this BAM file or you got it from someone. Sometimes people cap the maximum mapping quality to 50 because some downstream analysis softwares throw error if they find a mapping score of 255.

ADD REPLYlink modified 5.0 years ago • written 5.0 years ago by Ashutosh Pandey11k

I don't know why. The BAM file is got by myself, using the default tophat paramters. However, I have not found any side-effect so far. Thank you for your reply.

ADD REPLYlink written 5.0 years ago by zju.whw30

I've never fully trusted the mapping quality scores, so when I am interested in uniquely mapped reads I filter based on the SAM tags. I know that for TopHat v1.4.1 onwards (I haven't looked at earlier versions), the number of mappings for each read is reported in the "NH" tag, so you can write a simple script to only keep reads with this tag set to 1.

ADD REPLYlink written 5.0 years ago by Chris Cabanski320

Yes, you're right. There are at least two ways to get the uniquely mapped, NH:i:1 tag and mapping quality scores. I have made a mistake that I thought the NH:i:1 is in a constant column. But it is actually not. So it takes more time to identify the column number of the NH:i tag. And the bam file should be conversed to sam file, and converse back after filtering. On the other hand, you can use "samtools view -bq" to get the unique mapped reads. It is faster and easier. What is your scripts? What is your opinion

ADD REPLYlink written 5.0 years ago by zju.whw30

I agree that you can get uniquely reads quicker using samtools, assuming that you are confident in the mapping qualities. I was looking at metrics beyond counting/extracting the uniquely aligned reads, and I was using output from several different aligners, so I wrote a perl script for this task. This script is available as part of a software package at http://sourceforge.net/projects/rnaseqvariantbl/ (see extract_unique.pl).

ADD REPLYlink written 5.0 years ago by Chris Cabanski320

thank you very much

ADD REPLYlink written 5.0 years ago by zju.whw30
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