I have designed dual indexed primers (16S prok) with attached illumina adapters following protocoles from Kozich et al. (Schloss lab) and Takahashi et al. (2014). After miseq sequencing, i get sequences that are complementary to my adapters, starting from the 3' end of my primer. Basically, the sequencing went in the wrong direction. Do you know what could be the issue? I am guessing an inversion of read1 and read2 primers during the loading of the material, but i could be wrong. Thank you very much for your help!
Hi,
What was your insert size and for how many cycles was the Miseq-run run?
Hi,
What was the total number of reads? It's probable that your initial PCR was not efficient, if this is not the case, then It could be due to the size of your insert or number of Miseq run cycles -Was your Miseq-run in one direction(i.e 600bp )or in both directions (i.e 2x300)?
Hi Ashwini and Tolulope. thanks for your help. Insert size is around 450, which was checked on my initial PCR.At the end, I got 1,2 million reads, half of them were my barcoded specific primers, together with the 5' end adapter and some random nucleotides (with a length of 250bp). The other were the my reverse primer with the 3' end adapter (reverse compcomplemented) and random nucletodies (same thing 250 length). So the sequencing did work fine... I think read 1 and read 2 were inverted in the wells. could it be?
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