Hi, we recently get our data from Miseq PE300. We have 96 samples of bacteria V3-V4 region with the length of 630bp. The DNA library is built according the guide of illumina 16S metagenomic preparation guide. But instead of brought Nextera XT kit from illumina, we synthesis our index primers by invitrogen.
Our data shows that the average Q30 of both reads is very good, but the Q30 of i7 is relatively low (i7:28.2%,i5:92.3%) .
We thought their might be something wrong with the primers, so we checked all the primers by 3730 sequencing. The result shows there are some mismatches in the sequence but not in our index area. The oscillogram shows that the mismatch site always has double peaks. This may indicate that the primer is not pure enough. Both i7 and i5 primers have the same problems. So this might not be the reason why in Q30 of the i7 and 15 index so different.
Did anyone here encounter the same problem? What should we do to make things clear? Thank you for reading.
I have seen similar issues with low diversity libraries, particularly when doing 16s sequencing. How much phix/ control library do you use to balance the signal? I use >15% whether i'm using v2 or v3 chemistry and this gives a much higher Q30 for both reads.
Thank you for your help. We used 70% phix in our former run, which is much too high according to the advise of illumina tech support. So we changed our primers and reduce the proportion of phix to 40% this time. Hope we could get a better result this time.
We've got our results now. the Q30 of i7 index is 60.9% this time. but the Q30 of both reads decreased obviously. we decide to change to PE250 kits. Hope the results could be better.
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