Question: what read should i use for classifying?
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gravatar for life99945
11 weeks ago by
life999450
life999450 wrote:

Hi,

I have forward, reverse and extended illumina myseq 16s rRNA v4-v6 amplicon reads. Length of v4-v6 region is about 520-530 bp while my forward and reverse reads length is only 250 bp each. I checked all three of them in fastqc and it appears that forward read is the best. Should i use forward read for classifying or it is better to use an extended, even if it has worse quality.

p.s. How is it possible to get an extended file when forward and reverse reads are not enough long for crossing? And why extended read quality is so bad in the end? Does not the merged file should have a degradation in the middle?

Fastqc reports attached (i don't know how to add html file properly, so i just added these links, sorry for that):

forward: https://drive.google.com/open?id=1M3r47YJ8MrPMrY8FzwNOUZvB9dmZFL4o

reverse: https://drive.google.com/open?id=1z2Yb14Zf5H9PJtGqCebTelsigZS300au

extended: https://drive.google.com/open?id=1qw_zeKwqPyLEiiaRzolhltHK_tuFNAW6

Thank you!

ADD COMMENTlink modified 11 weeks ago • written 11 weeks ago by life999450

I've never used it, so I can't vouch for the results claimed in the paper, but in principle IM-Tornado uses information from both reads even if they don't overlap.

But how come the apparent mode of length for your extended file is 360bp, if the reads don't overlap?

edit: the quality of your reads in all files is very good and shouldn't impact much downstream analyses.

ADD REPLYlink modified 11 weeks ago • written 11 weeks ago by h.mon21k

Thank you for the answer! I'v got this extended file from laboratory along with forward and reverse reads, and i should ask them how they made it, but what if there is no big need in extended file? Maybe i can use only forward to classify my sample or it will increase the inaccuracy of the classification?

ADD REPLYlink modified 11 weeks ago • written 11 weeks ago by life999450
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