Hello together,
We would like to prepare barcoded fragment libraries of enzymatically digested genomic DNA. The fragment size range we are interested in is 40 to 240bp. We want to sequence 50bp using a SOLiD 4 machine.
My questions are:
1) What will happen if e.g. a 40bp fragment is sequenced? Will sequencing continue into the internal adaptor? There are various blocking oligos in the sequencing kit, and I am not entirely sure about their purpose. If they block the complete internal adaptor sequence, does this mean that only 34bp will be sequenced (I believe the fluorescent oligos are 8-mers, with 1st and 2nd base determining the color)?
2) Is it necessary to split the libraries into different sizes, e.g. 40-120bp and 120-240bp, to avoid PCR bias towards shorter sequences?
3) What else is there to take into account?
If anyone has experience on these questions, I would be happy if they could share. Thanks.
We would like to prepare barcoded fragment libraries of enzymatically digested genomic DNA. The fragment size range we are interested in is 40 to 240bp. We want to sequence 50bp using a SOLiD 4 machine.
My questions are:
1) What will happen if e.g. a 40bp fragment is sequenced? Will sequencing continue into the internal adaptor? There are various blocking oligos in the sequencing kit, and I am not entirely sure about their purpose. If they block the complete internal adaptor sequence, does this mean that only 34bp will be sequenced (I believe the fluorescent oligos are 8-mers, with 1st and 2nd base determining the color)?
2) Is it necessary to split the libraries into different sizes, e.g. 40-120bp and 120-240bp, to avoid PCR bias towards shorter sequences?
3) What else is there to take into account?
If anyone has experience on these questions, I would be happy if they could share. Thanks.
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