The question ermerged after reviewing this older application note by Illumina:
Their figure 1 indicates that the first position which is counted towards the targeted read length is the first base following the 3' end of the gene-specific primer.
Hence, the 515F-806R system targeting the V4 of the 16S rRNA gene is perfectly useable with a 2 x 150 base pairs MiSeq run, because of an 46 bp overlap within the 253 bp fragment covered between both gene-specific primers.
However, if one would assume that the first base counting for the target read length is the base following the sequencing primer, that obviously changes. So, if one would count in both primers, the overlap is reduced to <10 bases, even in the predictably worst part of boths read, qualitywise. Adding barcode(s) would even result in no overlap.
I hope this examples clarifies the question.
The underlying task is to find a primer pair that is feasible with MiSeq 2 x 250 with very good coverage and HiSeq 2 x 150 with lower coverage but higher yields.
I doubt that 2x150 bp HiSeq is a good system for hiseq. However, it seems that its OK to just use the forward read (according to caporaso et al 2011). What do you think?
Their figure 1 indicates that the first position which is counted towards the targeted read length is the first base following the 3' end of the gene-specific primer.
Hence, the 515F-806R system targeting the V4 of the 16S rRNA gene is perfectly useable with a 2 x 150 base pairs MiSeq run, because of an 46 bp overlap within the 253 bp fragment covered between both gene-specific primers.
However, if one would assume that the first base counting for the target read length is the base following the sequencing primer, that obviously changes. So, if one would count in both primers, the overlap is reduced to <10 bases, even in the predictably worst part of boths read, qualitywise. Adding barcode(s) would even result in no overlap.
I hope this examples clarifies the question.
The underlying task is to find a primer pair that is feasible with MiSeq 2 x 250 with very good coverage and HiSeq 2 x 150 with lower coverage but higher yields.
I doubt that 2x150 bp HiSeq is a good system for hiseq. However, it seems that its OK to just use the forward read (according to caporaso et al 2011). What do you think?
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