amplicon preprocessing vs.shotgun processing

[litali]

Hi,
what is the difference between the preproxessing to create sff files between amplicon and shotgun?

[flxlex]

Amplicons usually are very similar, and give brighter signals. So, besides having less fragments on a plate, also special algorithms for the basecalling are used.

[divinehammer]

Yeah amplicons are mostly similar so they either all light up or all go dark on a given dNTP flow, unlike shotgun frags which are heterogeneous in sequence

[MBS]

I did the analysis on our FLX-Cluster using the shotgun and amplicon setting on the same regions (was amplicon). This was the difference:

Analysed as shotgun:
Region Bases Reads AV_Len
3 26622782 116544 228.435
4 16631435 62620 265.593

Analysed as amplicon:
Region Bases Reads AV_Len
3 11555391 32334 357.376
4 12447187 36218 343.674

This is dramatic! Can someone explain this?

[kmcarr]

The amplicon filtering settings are much more stringent than the shotgun settings, explaining the increase in passed reads for the shotgun processed output. Additionally the amplicon processing does not permit any trimming of low quality bases from the 3' end of reads, essentially only accepting/rejecting reads in their entirety, whereas shotgun processing allows trimming of low quality bases at the 3' end to salvage the higher quality, 5' portion of a read. This explains the shorter average read length of the shotgun processed reads.