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Old 02-11-2016, 05:20 AM   #1
wri_ichp
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Location: Johnstown, PA

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Default Total RNAseq multiple mapping issue

We are running paired-end total RNA (using blood) on HiSeq and after adapter and quality trimming (Q>30), we use tophat2 for alignment. We are getting about 90% overall mapping, but around 80% of those are mapping to multiple locations.

I am pretty sure this is way too high, but don't know what the issue is. Are there any recommendations for how we can reduce the multi-mapping or ideas on what might have caused it. Thanks!
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Old 02-11-2016, 10:49 AM   #2
Brian Bushnell
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Mapping to a transcriptome will have a high rate of multiple mapping because the same references occur multiple times. If you want unique mapping, map to the genome instead, using a splice-aware aligner.

Also, trimming to Q30 will reduce the length of your reads and bias your data with no real advantage. Q10-12 will give you much better results. Short reads will multi-map more because they contain less information.
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Old 02-12-2016, 12:16 AM   #3
Michael.Ante
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I would rather guess, that you have rRNA contamination in your sample, or highly expressed transcripts originating from a cluster of similar genes. Check your reads with FastQC and if detected, blast your over-represented sequences. You can also use the repeat-masker to analyse your alignment towards rRNA and other elements.
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multi-mapping, multiple alignment, rnaseq alignment

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