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Hello everyone,
my institute recently purchased an Illumina MiSeq (Sep 2012). We want to do exome sequencing with the TruSeq exome capturing protocol, however we are not sure about the optimal way to design the experiment and prepare the libraries generating a maximal amount of high quality target data at lowest cost.
Since the standard kits from Illumina have either 50, 300 or 500 cycles, there are basically the six options to do paired end (PE) or single end (SE) sequencing with either kit. Now there are some considerations:
1. 2x250 PE or 500 SE will give the greatest output of sequenced bases (~ 7 GB). However since the fragment size is usually 350 bp, PE reads will overlap. Furthermore, the typical exon size is about 125 bp, so one will sequence many off target regions.
2. 2x150 PE or 300 SE will reduce the problem of overlapping reads and off-target regions, but also the output will be much lower (~ 4 GB). However, the cost is almost the same as in 1. The 300 cycle kit is slightly cheaper than the 500 cycle kit, but the fixed costs per run (e.g. the reagents) are the same, so one gets less for the same money.
We have not really considered to use the 50 cycle kit, since this is really short for exome sequencing. So my question is: What do you think is the best design for exome sequencing? 300 cycles or 500 cycles? PE or SE?
Thanks alot
my institute recently purchased an Illumina MiSeq (Sep 2012). We want to do exome sequencing with the TruSeq exome capturing protocol, however we are not sure about the optimal way to design the experiment and prepare the libraries generating a maximal amount of high quality target data at lowest cost.
Since the standard kits from Illumina have either 50, 300 or 500 cycles, there are basically the six options to do paired end (PE) or single end (SE) sequencing with either kit. Now there are some considerations:
1. 2x250 PE or 500 SE will give the greatest output of sequenced bases (~ 7 GB). However since the fragment size is usually 350 bp, PE reads will overlap. Furthermore, the typical exon size is about 125 bp, so one will sequence many off target regions.
2. 2x150 PE or 300 SE will reduce the problem of overlapping reads and off-target regions, but also the output will be much lower (~ 4 GB). However, the cost is almost the same as in 1. The 300 cycle kit is slightly cheaper than the 500 cycle kit, but the fixed costs per run (e.g. the reagents) are the same, so one gets less for the same money.
We have not really considered to use the 50 cycle kit, since this is really short for exome sequencing. So my question is: What do you think is the best design for exome sequencing? 300 cycles or 500 cycles? PE or SE?
Thanks alot