Different read lengths from dada2 denoising

[abdulg (Abdul Ghafar)]

G’day friends,

Thanks for taking time to answer my query. I am fairly new to qiime2 and using dada2 plugin in qiime2 for denoising my paired-end reads data. My target amplicon size is ~ 458 bp whereas I am getting features of varying lengths from 240 to 458.
I am using following command line:

qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux-V2.qza --o-table table-V2 --o-representative-sequences rep-seqs-V2-24030 --p-trunc-len-f 240 --p-trunc-len-r 240 --output-dir Denoised-V3

How can I avoid the shorter reads?

Thanks a lot

Abdul

[Nicholas_Bokulich]

HI @abdulg,

@abdulg

How can I avoid the shorter reads?

See here:

Filtering out ASVs from DADA2 based on length

Just wanted to toss out one more option for length-based filtering: qiime feature-table filter-seqs \ --i-data seqs.qza \ --m-metadata-file seqs.qza \ --p-where 'length(sequence) > 4' \ --o-filtered-data filtered-seqs.qza This works by viewing the FeatureData[Sequence] as Metadata, and then using a SQL-where clause to compute the length. The advantage here is then you don't have to export anything. This command above would keep any sequences longer than 4 nts.

Good luck!

[abdulg (Abdul Ghafar)]

Thanks a lot @Nicholas_Bokulich

Just for my knowledge, why are we getting shorter read lengths despite the fact that we are truncating both the forward and reverse reads. Theoretically it should discard anything shorter than that (in my case forwrad<240 and reverse<230). So I should not get such short reads like 300, 320 or so. Please give your insights. Thanks

[Nicholas_Bokulich]

@abdulg

why are we getting shorter read lengths despite the fact that we are truncating both the forward and reverse reads.

Because truncation happens before denoising and joining. So you are truncating to 240 nt, but then joining (merging) your reads and some are overlapping a lot, yielding a short read.