sequencing of short DNA fragments (40-240bp)

[volks]

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.

[pmiguel]

Originally Posted by volks

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?

Yes. That is what happens if you sequence short, bar-coded SOLiD amplicons. This used to be more of a potential problem during subsequent alignment to your reference sequence. But BioScope utilizes a seeded extension method that should limit the detrimental effects of adaptor sequence at the end of a read.

Originally Posted by volks

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)?

Not sure what you mean by "blocking oligo". SOLiD oligos are 9 bases, with the last 3 apparently being promiscuously pairing bases (eg, inosine) that stabilize associations made by the first 6 bases. Yes, the 1st and 2nd bases of the oligo specify the fluor attached to the oligo.

Originally Posted by volks

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?

Probably. Also, unless you are constructing these libraries yourself, the 40-120bp insert amplicons will likely be discarded during library size selection.

Originally Posted by volks

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.

Not exactly experience. But my suspicion would be that a mixture of very short amplicons with very long amplicons in a library will result in highly populated short amplicon beads and much more weakly populated long amplicon beads. The long amplicon beads may not even make it through enrichment. If they do, their signal will be much lower than that of the short amplicon beads. This will, at the least, probably cause sequence bias. At the worst, the bright, short amplicon beads might produce enough signal to bleed over signal from their weaker neighbors.

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Phillip