Hello! I am a quite new user of QIIME2 and I'm analyzing 6 samples of S.siderea (coral) looking for insights on the fungal communities. I used primers ITS-1F and ITS-2R and I worked with Illumina equipment. I will report on my pipeline in the hope that someone can help me figuring out my problem.
I am using Qiime qiime2-2022.2 version.
First I downloaded from UNITE the zip file "sh_qiime_release_16.10.2022.tgz" from UNITE in this link PlutoF DOI , then I followed this code to train a classifier :
Clean
awk '/^>/ {print($0)}; /[1]/ {print(toupper($0))}' sh_refs_qiime_ver9_99_16.10.2022.fasta | tr -d ' ' > sh_refs_qiime_ver9_99_16.10.2022_dev_uppercase.fasta
Import sequences
qiime tools import
--type FeatureData[Sequence]
--input-path sh_refs_qiime_ver9_99_16.10.2022_dev_uppercase.fasta
--output-path unite-ver9-seqs_99_16.10.2022.qza
Import taxanomy file
qiime tools import
--type FeatureData[Taxonomy]
--input-path sh_taxonomy_qiime_ver9_99_16.10.2022.txt
--output-path unite-ver9-taxonomy_99_16.10.2022.qza
--input-format HeaderlessTSVTaxonomyFormat
Train
qiime feature-classifier fit-classifier-naive-bayes
--i-reference-reads unite-ver9-seqs_99_16.10.2022.qza
--i-reference-taxonomy unite-ver9-taxonomy_99_16.10.2022.qza
--o-classifier unite-ver9-99-classifier-16.10.2022.qza
Up until this point I have no problems and I looked at my classifier file and it looks fine it is aprox 100, 000 MB and has plenty of rows with lines that have taxonomic information. The problem is in the next part, where I try to use my data with the classifier.
Import raw files of my samples (6)
qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path Raw --input-format CasavaOneEightSingleLanePerSampleDirFmt --output-path ../Run1/Qiime2/Run1.qza
Check Qscores
qiime tools view Run1.qzv
Quality score and number of reads:
Triming
nohup qiime dada2 denoise-paired --i-demultiplexed-seqs Run1.qza --p-trim-left-f 30 --p-trim-left-r 30 --p-trunc-len-f 250 --p-trunc-len-r 250 --p-n-threads 10 --output-dir dada_run1 --verbose &
classifier
qiime feature-classifier classify-skl
--i-classifier /home/xibalba/Sabina/UNITE/developer/unite-ver9-99-classifier-16.10.2022.qza
--i-reads representative_sequences.qza
--o-classification classified_seq.qza
Export file taxonomy.tsv
qiime tools export --input-path /home/xibalba/Sabina/Run1/Qiime2/dada_run1/classified_seq.qza --output-path /home/xibalba/Sabina/Run1/Qiime2/dada_run1/aln_tree
When I check on the file taxonomy.tsv I get this:
It appears as if the only classification I get is on the kingdom class, the results seem very wrong. Well thank you very much for your time and I hope someone can point me to the right direction on how to analyze my data.
^> âŠī¸