ASV table question of dada2 denoise

[wanjinhu]

Hi there:

When we finished dada2 denoise, we would get a ASV table. I am very confused about my results, because there is zero ASV shared in more than two samples, all ASV just appear in only one sample, which i really can't understand.

Script of what i run:

qiime tools import --type 'SampleData[SequencesWithQuality]' 
                   --input-format SingleEndFastqManifestPhred33V2 
                   --input-path manifest.tsv 
                   --output-path data_se.qza

qiime dada2 denoise-single --i-demultiplexed-seqs data_se.qza 
                           --o-table ASV_table-dada2.qza 
                           --p-trunc-len 0 
                           --o-representative-sequences rep-seqs-dada2.qza 
                           --o-denoising-stats stats-dada2.qza 
                           --p-n-threads 20 
                           --p-trunc-q 0 
                           --p-max-ee 2

qiime metadata tabulate --m-input-file ASV_table-dada2.qza 
                        --o-visualization ASV_table-dada2.qzv

Some files may help:
ASV_table-dada2.qzv (1.2 MB)
manifest.tsv (1.2 KB)

Here is a sub fastq of one sample A1:
A1_sub.fastq (951.8 KB)

Thanks for your help

wanjin.hu

[jwdebelius (Justine Debelius)]

Hi @wanjinhu,

I would make sure that you're trimming your primers and barcodes. If you still have barcodes attached to your sequences, it would result in unique ASVs.

I would recommend looking at q2-cutadapt; which is what I tend to use for this purpose.

Best,
Justine

[wanjinhu]

Yes, it worked after i trimmed adapter.

Thanks a lot