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Old 02-19-2014, 01:57 AM   #1
Junior Member
Location: India

Join Date: Feb 2014
Posts: 5
Exclamation Sorted bamfiles have increased size!!

I have RNA-Seq data from Illumina platform.
I aligned the reads post-QC onto hg19 bowtie index using tophat.
Now I wished to get the raw count of reads mapped to each gene using Ht-seq-count.

However the HT-Seq reports error that it is unable to find the mate pair and asks whether the sam file is properly sorted.
Hence, after reading posts on the problem. I sorted my bam files using samtoools.
samtools sort -n accepted_hits.bam accepted_hits_bam_sorted

Now the sorted BAM file has increased in size and I wish to know why his has happened.
Though it might not be but seems highly unintuitive.
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Old 02-19-2014, 02:26 AM   #2
Devon Ryan
Location: Freiburg, Germany

Join Date: Jul 2011
Posts: 3,480

That's not surprising. Remember that BAM files are compressed, with the compression algorithm being more efficient the more similar neighbouring reads are (i.e., coordinate-sorted files should compress to a greater extent).
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Old 02-19-2014, 02:37 AM   #3
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Location: India

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Thanks dpryan.
Just wanted to confirm if something went horribly wrong.
I'll now be following your earlier reply on a different post to run HTSeq
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Old 02-19-2014, 11:16 PM   #4
Peter (Biopython etc)
Location: Dundee, Scotland, UK

Join Date: Jul 2009
Posts: 1,543

You sorted by read name (-n), but the default sorting by mapping position puts similar reads together, and therefore their sequence data compresses well, so coordinate sorted BAM files are usually smaller.
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bam, htseq, rna-seq, samtools, tophat

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