problem on demultiflexing with paired-end fastq files

Hello, I am a student studying bioinformatics.

I'm getting a strange error while demultiflexing using a paired-end fastq file.

The quality plot graph keeps coming out strangely like this.


The command I used is:

qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path "./manifest.csv" --output-path ./demux.qza --input-format PairedEndFastqManifestPhred33

qiime demux summarize --i-data demux.qza --o-visualization ./demux.qzv

The contents of the manifest file :

sample-id,absolute-filepath,direction
sample1,$PWD/A21_S14_L001_R1_001.fastq.gz,forward
sample1,$PWD/A21_S14_L001_R2_001.fastq.gz,reverse
sample2,$PWD/A22_S15_L001_R1_001.fastq.gz,forward
sample2,$PWD/A22_S15_L001_R2_001.fastq.gz,reverse
sample3,$PWD/A23_S16_L001_R1_001.fastq.gz,forward
sample3,$PWD/A23_S16_L001_R2_001.fastq.gz,reverse
sample4,$PWD/A24_S17_L001_R1_001.fastq.gz,forward
sample4,$PWD/A24_S17_L001_R2_001.fastq.gz,reverse
sample5,$PWD/A31_S18_L001_R1_001.fastq.gz,forward
sample5,$PWD/A31_S18_L001_R2_001.fastq.gz,reverse
sample6,$PWD/A33_S19_L001_R1_001.fastq.gz,forward
sample6,$PWD/A33_S19_L001_R2_001.fastq.gz,reverse

how can i fixit?

Hello @crunchbro,

Welcome to the forums! :qiime2:

Those look like binned Q scores from the Illumina platform (PDF).

That's just how Illumina makes them these days. There's nothing to 'fix' :person_shrugging:

Are they causing you issues?

With this graph, it is difficult for me to know what length to cut in the step using dada2. Do you know how?

The quality looks high throughout, so I'm not sure if trimming is needed.

I did notice that the demux-joined plot shows reads to be 300 bp after joining, which implies little or no overlap. How long was the amplicon you sequenced? Maybe you could increase the overlap required to join so the output is the right length.

This is a file that sequenced the DNA of the frog's gut microbiota. I sequenced 96 samples at once, and the sequencing took 18-20 hours.

Cool! Did you sequence 16S V4 or 16S V3-V4 or another region? How long (in base pairs) do you expect the resulting amplicon to be?