We have given 59 samples to service provider for sequencing V1-V9 region using Miseq. With the demultiplexed data, we have started QIIME2 pipeline.
3.Total length of the amplicon
V1V3 500 ±10
V4V6 550 ± 10
V7V9 440 ± 10
I followed the following workflow for pair-end
- qiime tools import --type ‘SampleData[PairedEndSequencesWithQuality]’ --input-path pe-33-manifest --output-path paired-end-demux.qza --source-format PairedEndFastqManifestPhred33
- qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux.qza --p-trunc-len-f 280 --p-trunc-len-r 200 --o-representative-sequences rep-seqs.qza --o-table table.qza --o-denoising-stats stats.qza
After quality filtering, we lost more than 50% of sequence counts. This may be due to thw trimming parameter (not enough reads to merge).
Hence we tried with Forwards reads alone.
qiime dada2 denoise-single --i-demultiplexed-seqs single-end-demux.qza --p-trim-left 0 --p-trunc-len 280 --o-representative-sequences rep-seqs.qza --o-table table.qza --o-denoising-stats stats-dada2.qza --p-n-threads 4
In this , we could retain nearly 40% of the reads. I have attached table.qzv for your reference. I have also performed rarefaction curve
Alpha rarefaction curve: Observed OTUs, Shannon Index and faith pd
Sampling Depth: 30443
Retained Sequences: 1,552,593
Retained No.of samples: 51 out of 59
- Will the reads which I have got after filtering be sufficient enough for the taxonomic classifications in the publication point of view?
- Can I take this results as authenticated one?
- Will this sequence counts are good enough?
- Is there any publications to support our sequence counts? What is the minimum sequence counts for a sample?
- Are my rarefaction curve and sampling depth good?
Kindly help. We are at the crucial point to decide to go on or not?
alpha-rarefaction_30443.qzv (461.2 KB)