number of cycles, Nextseq500, for whole exoems equencing, which option is more efficient?

Question

[lait]

Hi all,

we are moving from Illumina Hiseq2500 to Nextseq500 soon. For our Hiseq2500 we had reads of 101 bp length. We mainly use it for whole exome sequencing, and we have a well established pipeline for the full analysis of the WES data.

-1-

• With respect to Nextseq500, my question is: could you provide me with suggestions on the number of cycles to choose between 75, 150 and 300? as mentioned, we mainly need it for whole exome sequencing.

• did anyone used the Nextseq500 for WES with 75 bp reads length? was it efficient ? or should we go for longer reads?

• any additional parameters should be taken into consideration when choosing the number of cycles?

-2-

I see no major changes should be applied to the already existing whole exome sequencing data analysis pipeline when switching from Hiseq to Nextseq, do you have any comments on this point? we are planning to do some benchmarking here and resequence some samples with nextseq which were already sequenced with hiseq, and compare the output.

Thanks in advance for your input!

(P.s.:with respect to depth, we aim for 60x as a minimum, thats why we used to sequence each sample twice when using Hiseq2500 which used to give us around 30x depth per sample.)

(P.s.2: this is a bioinformatics-related question and not a wet-lab one :) I want to know the impact of the number of cycles (read length) on our WES analysis.)

[GenoMax]

we are moving from Illumina Hiseq2500 to Nextseq500 soon. For our Hiseq2500 we had reads of 101 bp length. We mainly use it for whole exome sequencing, and we have a well established pipeline for the full analysis of the WES data.

If you are going to compare results across samples then you would need to contend with a batch effect. If you also change the length of reads that would be another variable to consider. You are also changing chemistries from 4-color to 2-color. Good to see that you are thinking about the effect this change will have on results :

we are planning to do some benchmarking here and resequence some samples with nextseq which were already sequenced with hiseq, and compare the output.

Answer 1

[trausch]

Usually the insert size (fragment size) is small for WES. To avoid a lot of overlapping reads that are not independent observations we usually sequence only 2x75bp on a NextSeq.

Answer 2

[Tm]

I agree with trauch. But, mostly we sequence it using 2x150 bp chemistry and it gives good results. However, I think there is no reason of using 2x300bp chemistry for exome.