Hello, everyone.
I'm trying to use DADA2 to work with paired-end sequences of 16S V3-V4 region (338F/806R) from soil samples (Illumina MiSeq). I have removed primers and barcodes from the sequences from q2-cutadapt. And here are the quality plot of my sequences.
B_trimedSeq.qzv (319.8 KB)
After that, I run dada2 to denoising the sequences. For the extremely poor quality of reverse reads, I tried to trim more in the reverse length, but I need to ensure enough overlap region for merging. So I trimmed the length of 272 for forward reads, and 231, 217 for reverse reads.
Here are my commands:
time qiime dada2 denoise-paired \
--i-demultiplexed-seqs ./A_trimedSeq.qza \
--p-trim-left-f 0 \
--p-trim-left-r 0 \
--p-trunc-len-f 277 \
--p-trunc-len-r 203 \
--p-n-threads 20 \
--o-table ./DADA2/dada2_Tab.qza \
--o-representative-sequences ./DADA2/dada2_Reps.qza \
--o-denoising-stats ./DADA2/dada2_Stats.qza
--verbose
Here are some outputs:
1) Filtering The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C1_0_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C1_126_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C110_109_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C110_235_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C122_121_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C122_247_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C24_23_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C24_149_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C30_29_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C30_155_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C53_52_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C53_178_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C7_6_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C7_132_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C47_46_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C47_172_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C41_40_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C41_166_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C70_69_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C70_195_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C64_63_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C64_189_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C76_75_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C76_201_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpl9lu_p1_/filt_f/C99_98_L001_R1_001.fastq.gz and /tmp/tmpl9lu_p1_/filt_r/C99_224_L001_R2_001.fastq.gz not written.
Some input samples had no reads pass the filter.
By the way, I use QIIME2 2021.4 installed by conda in a linux platform.
I've read similar posts in our formula, most of which suggested to trimmed strictly for sequences length. However, it seemed not to work for me. So I upload my questions and results here and ask for help. I'm so depressed now, and Can anybody give me some suggestions? Thank you very much in advance.
Sirong