Hi,
I have a variable length sequence cloned between two known sites, and I want to do paired-end reads and accurately count the number of times each sequence occurs (kind of like digital gene expression but for DNA). Ideally I'd like to do this for all sequences between 100-1000bp, and to not have strong insert length biases in my counting. I get the impression that this is impossible. So, I'm wondering two thigs:
1) how long can I go for inserts on the MiSeq and/or HiSeq2500 ?
2) how will the efficiency of cluster generation / sequencing quality be effected by insert-length, and could I correct for decreased efficiency at long lengths by generating some sort of a curve in which I do a sequencing run with known amounts of inserts of various sizes and sequence complexity?
I know I can amplify, shear, and ligate adapters, but I fear that this may give less quantitative read counts.
thanks for any info you can provide
-Lucas
I have a variable length sequence cloned between two known sites, and I want to do paired-end reads and accurately count the number of times each sequence occurs (kind of like digital gene expression but for DNA). Ideally I'd like to do this for all sequences between 100-1000bp, and to not have strong insert length biases in my counting. I get the impression that this is impossible. So, I'm wondering two thigs:
1) how long can I go for inserts on the MiSeq and/or HiSeq2500 ?
2) how will the efficiency of cluster generation / sequencing quality be effected by insert-length, and could I correct for decreased efficiency at long lengths by generating some sort of a curve in which I do a sequencing run with known amounts of inserts of various sizes and sequence complexity?
I know I can amplify, shear, and ligate adapters, but I fear that this may give less quantitative read counts.
thanks for any info you can provide
-Lucas