22 August 2013

Aphaenogaster, RNAseq, transcriptome, simulation, fastq, and git

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Thursday 22 August



Simulation running through oases! For 12 input Arabidopsis mRNA. Stats for merged assembly (K=25) after individual assemblies from 19 to 25, after digital normalization to 20:

Total Contigs 33
Total Trimmed Contigs 33
Total Length 28105
Min contig size 408
Median contig size 644
Mean contig size 851
Max contig size 2302
N50 Contig 11
N50 Length 918
N90 Contig 28
N90 Length 580

Note that longest transcript in test file is 2,313bp so max contig from assembly looks quite close!

Now following guide to Counting reads for RNA-seq with more info on bwa here

Success! First column is transcript, second and third columns are the stard and end of each transcript that reads could map to (all start at 0 and span entire length of transcript). The fourth column is the read counts in the bam file. Reads map to each sample!

ENA|AAA16571|AAA16571.1 0   558 10622
ENA|AAA20048|AAA20048.1 0   783 10876
ENA|AAA21761|AAA21761.1 0   933 4454
ENA|AAA32754|AAA32754.1 0   2313    87408
ENA|AAA32773|AAA32773.1 0   594 2122
ENA|AAA32782|AAA32782.1 0   1152    5452
ENA|AAA32797|AAA32797.1 0   1116    18198
ENA|AAA32815|AAA32815.1 0   855 21690
ENA|AAA32838|AAA32838.1 0   969 10662
ENA|AAA32852|AAA32852.1 0   613 10306
ENA|AAA32864|AAA32864.1 0   918 1938
ENA|AAA32895|AAA32895.1 0   447 8768

Compare to the simulated read counts from rlsim, where the value after the $ is the simulated expression level:


Hmmm…NO correlation between known and estimated gene expression levels (r = -0.12).

<img src="http://www.johnstantongeddes.org/assets/img/sim-GE-plot.png" alt="plot" width="300" height="300">



Debugged and ran complete velvet-oases for A22 assembly after digital normalization and combining contigs with FLASH, only required ~30 minutes and 15GB RAM.

Stat using khmer assemstats3.py script

** cutoff: 0

N sum max filename
90612 86485229 14006 transcripts.fa

** cutoff: 300 N sum max filename —— ——— —— —————– 72334 81983476 14006 transcripts.fa

Given my expectations for transcriptome size of 17,000 genes with typical length of 1000 bp = 1.7 × 107 I’m getting on the same order of magnitude, but 4.8226-fold more, which is to be expected given alternative splicing!

And using the /khmer/sandbox/assemstats2.py script,

Total Contigs           90612
Total Trimmed Contigs   72334
Total Length            81983476
Min contig size         300
Median contig size      765
Mean contig size        1133
Max contig size         14006
N50 Contig              14983
N50 Length              1598
N90 Contig              51215
N90 Length              500

To be continued…


Moyers, B.T. & Rieseberg, L.H. (2013). Divergence in Gene Expression Is Uncoupled from Divergence in Coding Sequence in a Secondarily Woody Sunflower. International Journal of Plant Sciences, 174, 1079–1089.


Great tip to use trash-put in the trash-cli program for Recycle Bin functionality when deleting files commandline. Do not use an alias for rm as many scripts use this to clean up temporary files that I really do not want saved.

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