Chicken/Non canonical animal sequencing depth and read length

Question

[Rafael Soler]

Hi!

Do you know what is the correct Illumina sequencing protocol to perform?

We are between 50 bp Hiseq 2500 with a sequencing depth of 50M of reads, or NexSeq 75-150 bp read length with a sequencing depth of 30M.

What is more important for non canonical organisms (like chicken and others) whom genome is not very well annotated? Longer reads (More unique mapping reads) or more sequencing depth althought the read whould be smaller?

Thank you!!

[ATpoint]

Depends what you want to do. If you are after de novo assembly then probably longer reads are preferred. For standard DE testing the normal procedures as used in any other organism should apply. If this is for DE testing I personally would prefer longer reads since 30M is still plenty for "normal" RNA-seq. I personally aim for 25M per sample in a standard DE experiment in human/mice. Read depth is not too much a factor towards statistical power as long as you are not very low so technical dropouts would be a factor, replicate number is far more important.

[GenoMax]

Consider recommendations from Illumina on this topic. It should not much matter even if you are working with a not so "complete" genome even though chicken genome is reasonably well complete. What ever combination you choose make sure you include biological replicates. More the better but at least 3 if you can.