Good morning,
I'm getting poor results in my analysis: about 500 ref seqs and only 30% of them are matched in taxonomy assignment. How can I obtain more ref seqs/ASV and improve the matching ratio? What if I remove "--p-discard-untrimmed" option from trim command? Thank you in advance!
I start from 20 FASTQ, paired end Illumina, 300 bp.
There are the commands I entered in order:
qiime cutadapt trim-paired --i-demultiplexed-sequences demux-paired-end.qza --p-adapter-f ' CCTACGGGNGGCWGCAG... GGATTAGAWACCCBNGTAGTC ' --p-adapter-r ' GACTACNVGGGTWTCTAATCC... CTGCWGCCNCCCGTAGG ' --p-cores 4 --o-trimmed-sequences trimmed-seqs.qza --p-discard-untrimmed --verbose
=== Summary ===
Total read pairs processed: 58,375
Read 1 with adapter: 50,754 (86.9%)
Read 2 with adapter: 48,590 (83.2%)
Pairs that were too short: 82 (0.1%)
Pairs written (passing filters): 43,664 (74.8%)
qiime demux summarize --i-data trimmed-seqs.qza --o-visualization demux.qzv
qiime dada2 denoise-paired --i-demultiplexed-seqs trimmed-seqs.qza --p-trim-left-f 0 --p-trim-left-r 0 --p-trunc-len-f 284 --p-trunc-len-r 236 --o-table table.qza --o-representative-sequences rep-seqs.qza --o-denoising-stats denoising-stats.qza
qiime feature-classifier classify-consensus-vsearch --i-query rep-seqs.qza --i-reference-reads silva-138-99-seqs.qza --i-reference-taxonomy silva-138-99-tax.qza --p-perc-identity 0.99 --p-threads 4 --o-classification assigned_taxa.qza –verbose
Matching query sequences: 162 of 526 (30.80%)
demux.qzv (321.1 KB)