![]() ![]() This is why for example DADA2 by default has a truncQ of 2 and I’ve personally never needed to change that. With these newer methods however, they attempt to correct these base-calls and so do not rely on a min Phred-score thresholds, within reason. Before denoising methods such as DADA2/Deblur/Unoise came out, that was necessary as one means of ensuring we weren’t introducing too much error by allowing low quality reads. The min Phred score 20 that you mentioned indeed was a unofficial standard in the field when we were working with OTU clustering methods. I’ll start by the conclusion which is to say what you are seeing is perfectly normal and nothing unexpected is happening. And thanks for sharing your results and searching around on the forum as well. It’s great to see users diving so deep into this stuff because it really does highlight how much expertise goes into these analyses and how specific each analysis can be. I calculated for all the % of reads that passed denoising. What is the reasonable % of reads that pass good filtering (if the sequencing is good quality offcourse)? Could you confirm this? All in all the result is similar to forward read only denoising, -p-trunc-q 20 Here, 'click' it was said that if they are not similar it may mean I have singletons. deblur denoising showed that reads-raw and reads-derep vary a lot.In other option I send you here I think it may be a problem with merging of the read so I proceeded with analysis of only forward reads.I dont think yet it is a main reason for such a huge read loss. Yet I always thought that 20 is some kind of a threshold that at first I should check. what and mentioned - it may be one of reasons to decrease the -p-trunc-q value to default version.o-representative-sequences rep-seqs-deblur.qza p-trunc-q 10 stats-dada2_LB18_31-R1-10.qzv (1.2 MB)Īdditionally, I counter proof all above I used another method for denoising deblur in case of forward reads only Chimera filtering stayed 'consensus' in all Result: stats-dada2_LB18_31-R1-noQS.qzv (1.2 MB)Īlso, I did with another Phred score selection. ![]() o-denoising-stats stats-dada2_LB18_31-R1-noQS.qza o-representative-sequences rep-seqs-dada2-LB18_31-R1-noQS.qza i-demultiplexed-seqs FastQ-LB18_31-demux-R1.qza o-denoising-stats stats-dada2_LB18_31-nochim.qza o-representative-sequences rep-seqs-dada2-LB18_31-nochim.qza o-denoising-stats stats-dada2_LB18_31-default.qza o-representative-sequences rep-seqs-dada2-LB18_31-default.qza i-demultiplexed-seqs FastQ-LB18_31-demux.qza
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