10 October 2013

transcriptome, TPM, BWA, and simulation

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de novo gene expression mapping works!

Gene expression counts

With BWA, have actual read counts, but need normalized counts - Reads per Kilobase per Million mapped reads (RPKM) typically reported but recently shown that this is problematic for comparison among samples so Transcripts per Million (TPM) is endorsed (Wagner et al. 2012). Bizarrely, couldn’t find any scripts to do this from BWA and the original paper not clear on how to do so. Fortunately found great blog post with a nice worked example that I was able to recreate.

Made my example available as a gist

Useful presentation on TPM here

Success!!! Working with TPM instead of raw gene counts results in a strong correlation (r = 0.90) between known expression and TPM from mapping reads to Oases transcriptome. Resolves issue from first round of gene expression simulation.

Correlation between known and mapped reads for gene expression data
Correlation between known and mapped reads for gene expression data

Now that I know it works and have a functioning pipeline, proceed to full analysis!

BWA mapping

When I map reads to the known transcripts, results are as such

Locus Start Stop Count
ENA|AAA02840|AAA02840.1 0 1092 18584
ENA|AAA03449|AAA03449.1 0 765 7458
ENA|AAA16569|AAA16569.1 0 504 5984
ENA|AAA16570|AAA16570.1 0 522 7236
ENA|AAA16571|AAA16571.1 0 558 14094

where the first column is the gene, second and third columns are start and stop locations where reads were aligned to, and fourth column is the number of reads mapping to that transcript.

However, when I map reads to Oases assembled transcripts, I get NO mapped reads

Locus Start Stop Count
Locus_1_Transcript_1/10_Confidence_0.450_Length_1487 0 1487 0
Locus_1_Transcript_2/10_Confidence_0.325_Length_1543 0 1543 0
Locus_1_Transcript_3/10_Confidence_0.550_Length_1398 0 1398 0
Locus_1_Transcript_4/10_Confidence_0.525_Length_1705 0 1705 0
Locus_1_Transcript_5/10_Confidence_0.525_Length_1401 0 1401 0
Locus_1_Transcript_6/10_Confidence_0.450_Length_1669 0 1669 0

Odd. Maybe because of multiple isoforms in Oases assembly..though I thought it tried to take them out by ‘popping bubbles’

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