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Old 10-05-2016, 12:48 PM   #1
liumangmang
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Default human RNA seq alignment QC

Hi,
I used HISAT2 to align human RNA-seq data (Illumina PE, Stranded, rRNA depleted).
We are interested in comparing gene expression levels, snp and different isoform usage between treatment conditions in the end.
I got about 70-75% reads aligned concordantly 1 time and about 20% concordantly >1 time with less than 10% disconcordantly aligned. Overall mapped rate is over 95%.
Is there a common benchmark that how much % unique concordantly mapped reads is typical for human RNA?
I'm wondering how to determine whether alignment result is acceptable for carrying on the downstream analysis and how I could tweak with the parameters in HISAT if needed.....

Thank you,

Last edited by liumangmang; 10-05-2016 at 12:59 PM.
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Old 10-06-2016, 12:21 AM   #2
SylvainL
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Sounds good to me... Over 70% of uniquely mapped reads for Human is quite good. You also have to keep in mind that this percentage can obviously depend on the expressed genes...
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Old 10-06-2016, 01:24 AM   #3
Michael.Ante
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Hi,

Before alignment I'd perform QC with FastQC and FastQC screen (especially in case of rRNA depletion).
After alignment, I'd perform QC with a set of RSeQC's tools:
  • bam_stat.py
  • clipping_profile.py
  • geneBody_coverage.py
  • infer_experiment.py (if your library was strand-preserving)
  • inner_distance.py (for PE-runs)
  • read_distribution.py

A lot of results can be collected by the multiQC reporting tool.

Cheers,
Michael
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Old 10-06-2016, 01:33 AM   #4
Persistent LABS
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Hi liumangmang,
Your alignment summary looks good. There is a similar post here: http://seqanswers.com/forums/showthread.php?t=29769
You can refer this.
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Old 10-06-2016, 07:13 AM   #5
liumangmang
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Thank you all, I'll try those QC tools and come back to update.
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Old 11-15-2016, 01:19 PM   #6
liumangmang
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Update:
We get average about 15%-25% multi mapped reads. And there are some public available data from SRA (same tissue type and protocal), also get similar multi mapped ratio.
And RSeQC + multiQC really worked well, it gave me pretty summary report!

Thank you all.
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