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Old 07-29-2011, 06:39 AM   #1
Location: 02119

Join Date: Jul 2011
Posts: 29
Default base composition and base calling

I've read that the illumina basecalling software has problems calibrating itself if the base composition in the first few bases of the reads isn't a roughly equal mix of nucleotides. We're thinking of sequencing constructs that begin with our own barcode and were wondering what the parameters are for correct base calling:

- how many positions are used to calibrate?
- what are the bounds on acceptable nucleotide mixture? eg, how far off from 25% each can you be?
- I believe you can calibrate on something other than the first four bases. How far into the read can you wait to calibrate?
- can different lanes in a run be calibrated differently? eg, if our sample is one lane of a run, does that make this easier or harder for the sequencing facilitiy?
- does any of this vary between the GAII and HiSeq?

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