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  • Bisulphite sequencing on Illumina Paired End 100bp reads

    Hi All Im new on here. i was after advice concerning the 100bp PE reads for bisulphite sequencing:

    I would like to sequence directly from 200bp PCR fragments as I am hoping to index 96 samples in a single lane.

    Is it possible to sequence many (~150) PCR amplicons that are all 200bp long using PE 100bp. I was hoping to generate similar sized bisulphite PCR amplicons (~200bp) so there would be no need for size selection and this is a reliably obtainable size for bisulphite PCR.

    Also I really have no other option but to use PCR products as I plan to do bisulphite sequencing and my source material would be bisulphite PCR products. These bisulphite PCR amplicon sequences are to be aligned to a small genomic region (a large gene) where all my amplicons will come from (300kb region of bisulphite converted sequnce, which will be used specifically for the alignment) and some of these 200bp amplicons will overlap with one another (say where I was interested in a streach of 3kb or so and covered it with a number of 200bp PCR amplicons).

    In essence I want as much read length (100bp x2) from the 200bp PCR amplicons as possible to look at methylated cytosines and SNPs throughout the full length of the same amplicon and so my thinking is there will be no insert for the 100bp PE read as I want data on the whole (or as much as possible) of the 200bp PCR amplicon - is this possible to achieve?

    Also I have heard it is a problem bioinformaticaly that if you do 100bp PE reads, you ideally don't want the reads from either end to overlap (ie for the sequncing from either end inwards) coz if they do you can't align these easily or you get over represented sequnces?

    Thanks guys

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