unequal ratio of C and G in RNAseq

[jtamames]

Dear friends

We have some data of Arabidopsis transcriptomics. The quality of the sequencing looks good, but I am puzzled by the different ratio of G and C all along the sequences, as you can see in the attached figure. As we are sequencing cDNAs, shoudn´t we expect equal ratios for G and C?

Any hint will be very appreciated. Thanks!

[Markiyan]

Did your's illumina system use 4 channel or 2 channel imaging?

So did yours illumina system use 2 channel (like nextseq) or proper 4 channel imaging (like Miseq?).

This type of trace reminds me of the nextseq RNAseq results, where G has the same meaning as no signal, so if the read signal drops too low after G, than it will be trimmed with that G. So it would cause similar bias to what you had observed.

Personally I like 4 channel imaging more esp for de novo work, due to 2-3 times lover error rate, more robust dephasing calculations and longer reads, but for RNAseq it is less critical in most cases.

[jtamames]

Hello Markiyan

Thanks for answering. I forgot to say, it is Hiseq sequencing, so it is 4-channel imaging

[kmcarr]

Dear friends

We have some data of Arabidopsis transcriptomics. The quality of the sequencing looks good, but I am puzzled by the different ratio of G and C all along the sequences, as you can see in the attached figure. As we are sequencing cDNAs, shoudn´t we expect equal ratios for G and C?

Any hint will be very appreciated. Thanks!

Your sequencing libraries were almost certainly constructed using a strand specific preparation kit. Since these libraries maintain strand orientation, as opposed to randomly oriented ds-cDNA fragments, the composition biases which are naturally present in RNA coding strands are maintained. This plot is very normal for Arabidopsis RNA.