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Thread | Thread Starter | Forum | Replies | Last Post |
Forcing paired-end data mapped as single-end in SAM | puggie | Bioinformatics | 1 | 03-16-2013 10:50 AM |
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Does Cufflinks support single-end and paired end data together ? | ersenkavak | Bioinformatics | 1 | 10-22-2010 07:26 AM |
Paired-end Illumina data | mchaisso | Bioinformatics | 7 | 07-17-2008 11:52 AM |
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#1 |
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Location: USA Join Date: Dec 2011
Posts: 45
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Hello,
I have aligned my RNA-Seq paired-end data to a reference using Bowtie2: Bowtie2 -x Ref -1 pair1.fq -2 pair2.fq -S out.sam In order to calculate RPKM we need to know the total number of mappable reads in the experiment which was obvious in the case of single end data. I am confused now which number to use as "mappable reads in the experiment" in the case of paired-end data. This is Bowtie2 alignment summary 19231140 reads; of these: 19231140 (100.00%) were paired; of these: 1790875 (9.31%) aligned concordantly 0 times 4367667 (22.71%) aligned concordantly exactly 1 time 13072598 (67.98%) aligned concordantly >1 times ---- 1790875 pairs aligned concordantly 0 times; of these: 30148 (1.68%) aligned discordantly 1 time ---- 1760727 pairs aligned 0 times concordantly or discordantly; of these: 3521454 mates make up the pairs; of these: 2645377 (75.12%) aligned 0 times 322397 (9.16%) aligned exactly 1 time 553680 (15.72%) aligned >1 times 93.12% overall alignment rate Thank you in advance! |
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#2 |
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Location: Oxford, UK Join Date: Nov 2011
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So to get 'mappable reads' I would look at the first output which is the read stats.
--- (1) 19231140 reads; of these: (2) 19231140 (100.00%) were paired; of these: (3) 1790875 (9.31%) aligned concordantly 0 times (4) 4367667 (22.71%) aligned concordantly exactly 1 time (5) 13072598 (67.98%) aligned concordantly >1 times --- So in a plain English: Out of 19231140 reads (1) all where paired (2), 1790875 did not align (3) and 17440265 (4+5) aligned 1 or more times. therefore 17440265 reads were mapped. The next 2 outputs just explain why the unmapped reads (3) didn't align I guess. Hope that helps. |
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#3 |
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Location: USA Join Date: Dec 2011
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Thanks David, I have been looking for tools to analyze the output file from bowtie2 (SAM formatted) and found a couple (samtools flagstat and RSeQC):
The output from samtools flagstat is: in total (QC-passed reads + QC-failed reads) 38462280 + 0 duplicates 0 + 0 mapped (93.12%:-nan%) 35816903 + 0 paired in sequencing 38462280 + 0 read1 19231140 + 0 read2 19231140 + 0 properly paired (90.69%:-nan%) 34880530 + 0 with itself and mate mapped 35157518 + 0 singletons (1.71%:-nan%) 659385 + 0 with mate mapped to a different chr 174452 + 0 with mate mapped to a different chr (mapQ>=5) 45969 + 0 you can see that the overall alignment reported by bowtie2 is what samtools flagstat called "mapped (93.12%:-nan%) 35816903 + 0". Now, I am not sure wither to add the singletons (read pairs in which only one tag is successfully mapped) to the "mapped" or not, in order to use it to calculate RPKM |
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#4 | ||
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Location: Oxford, UK Join Date: Nov 2011
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Yes I have tried those packages before. I also like Picards tool set for alignment metrics such as
Quote:
I guess the question to ask would be weather you could change tack a little and use FPKM. Take a look at what cufflinks says here Quote:
http://seqanswers.com/forums/showthread.php?t=10332 David |
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#5 |
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Location: USA Join Date: Dec 2011
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Thanks Daivd, That was helpful. I guess I am going to try Cufflinks and see how it goes. As I already have the SAM/BAM files, so it should be easy to try it.
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#6 |
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Location: Oxford, UK Join Date: Nov 2011
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No worries,
This is a good paper to help with analysis using cummbeRbund also. http://www.nature.com/nprot/journal/....2012.016.html |
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#7 |
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Location: USA Join Date: Dec 2011
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Excellent, I will look at it as well
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