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Old 09-16-2014, 08:09 AM   #1
sbdk82
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Default HTSeq warning message

I am using HTSeq-count to count paired end reads (2 samples with 3 replicates in each) . But I am getting the warning message which says "some reads with missing mates encountered". Am I doing something wrong? Following are the missing mates in all 6 reads.

HTML Code:
Warning: 29572652 reads with missing mate encountered.
79486404 SAM alignment pairs processed.

Warning: 29467379 reads with missing mate encountered.
74028848 SAM alignment pairs processed.

Warning: 41994492 reads with missing mate encountered.
108334368 SAM alignment pairs processed.

Warning: 31994985 reads with missing mate encountered.
81980266 SAM alignment pairs processed.

Warning: 145791964 reads with missing mate encountered.
150324577 SAM alignment pairs processed.

Warning: 128675855 reads with missing mate encountered.
132292695 SAM alignment pairs processed.
I am using the following command
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htseq-count -r name -m intersection-strict -s no -i gene_id  <sorted_by_name.sam> <GTF File>   >  <Count File>
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Old 09-16-2014, 09:20 AM   #2
dpryan
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Which aligner did you use? Odds are good that these are singletons.
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Old 09-16-2014, 09:30 AM   #3
sbdk82
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I used BWA-MEM for alignment. Then used samtools to sort the SAM file using this

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samtools view -b -S File.SAM > File.BAM
samtools sort -n File.BAM   File_sorted
samtools view -h  File_sorted.bam > File_sorted.sam
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Old 09-16-2014, 09:37 AM   #4
dpryan
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What happens if you:
Code:
samtools view -SF 8 File_sorted.sam | htseq-count -r name -m intersection-strict -s no -i gene_id  - <GTF File>   >  <Count File>
If the warnings go away, then you know that this is due to singletons. With bwa mem, this could also be due to chimeric/fusion/non-linear alignments. I don't use bwa mem with RNAseq datasets, so I've not thought much about how such alignments might get treated by htseq-count.

BTW, you can skip the sorting and conversion to/from BAM and just use the initial SAM (or BAM if you pipe bwa mem to samtools) file. You don't have to actually name-sort things, the aligner will output pairs together anyway and that's all that htseq-count wants.
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Old 09-16-2014, 09:45 AM   #5
sbdk82
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Thanks !! I will try that.

I used the original SAM as you suggested in an earlier post, but that gave me some error. So I tried with sorted sam. I am using BWA-MEM because tophat2 cannot handle the large files. Can you please suggest any other aligner for RNA-Seq reads?

I am trying STAR aligner now, but getting segmentation fault while mapping the reads

<HTML>./STAR --genomeDir /PATH/index/ --readFilesIn /Read_PATH/_L001_R1.fastq, /Read_PATH/_L002_R1.fastq /Read_PATH/_L001_R2.fastq, /ReadPATH/_L002_R2_001.fastq --runThreadN 10<HTML>
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Old 09-16-2014, 09:50 AM   #6
dpryan
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I was about to suggest using STAR, it's what I use. Which version of STAR are you using? I know that some people have reported segfaults of some of the recent versions (btw, you should post that to the user forum, Alex Dobin is one of the few aligner authors who actually provides timely feedback), though I've not had any issues myself.
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Old 09-16-2014, 10:00 AM   #7
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I am using the latest version(2.3.0.1) . Yes, I saw some other people are also getting seg fault. I will post that on google group. Anyway, Thanks a lot for your help
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Old 09-16-2014, 10:02 AM   #8
dpryan
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It's actually up to 2.4.0c, but you'll have to get it from github now.
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Old 09-16-2014, 10:08 AM   #9
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I see. Thanks !!
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