72bp Illumina GAII reads on small RNA library.
The libraries checked out at ±110bp by PAGE, with a bit of a smear upwards from here. I gel-purified between 100 and 150 bases from the gel.
Two main problems:
a) The vast majority of the reads do not contain a small RNA insert. The starting material was low (50-400ng) total RNA input, but given that the library size looked good, I was hoping for better.
b) The reads tend to reach the end of the known library sequence, and are then followed by runs of As, and then random nucleotide sequence. The quality scores are low in this region, but is this common for readthrough beyond the end of the library? I would have expected Ns.
Can anyone suggest reasons why the libraries looked to be of appropriate length, but there were no inserts? Are runs of sequence beyond the end of the library product 'real' polynucleic acid, and can they give the false library sizes? Is there a way to remove them.
Thanks for any suggestions...
The libraries checked out at ±110bp by PAGE, with a bit of a smear upwards from here. I gel-purified between 100 and 150 bases from the gel.
Two main problems:
a) The vast majority of the reads do not contain a small RNA insert. The starting material was low (50-400ng) total RNA input, but given that the library size looked good, I was hoping for better.
b) The reads tend to reach the end of the known library sequence, and are then followed by runs of As, and then random nucleotide sequence. The quality scores are low in this region, but is this common for readthrough beyond the end of the library? I would have expected Ns.
Can anyone suggest reasons why the libraries looked to be of appropriate length, but there were no inserts? Are runs of sequence beyond the end of the library product 'real' polynucleic acid, and can they give the false library sizes? Is there a way to remove them.
Thanks for any suggestions...