I am trying to run qiime deblur denoise-16S on 16S V3-V4 amplicon data (NovaSeq PE250), but I keep encountering the following error during the MSA step:
msa failed for file.fastq.gz (maybe only 1 read?)
Attempted fixes:
- Checked sequence quality with
demux.qzv(looks normal). - Adjusted
--p-trim-length(tried 120, 150, 200, 400) – same error persists. - Modified
--p-min-reads and --p-min-size parameters – no improvement.
Environment:
- QIIME2 2024.10
- Command used:
#!/bin/bash
mkdir -p ~/exportone
echo -e "#SampleID\tabsolute-filepath\tdirection" > ~/exportone/manifest.tsv
for file in ~/gutfastq/*.fastq.gz; do
sample_id=$(basename "$file" .fastq.gz | tr '_' '-')
echo -e "${sample_id}\t$(realpath "$file")\tforward" >> ~/exportone/manifest.tsv
done
qiime tools import \
--type 'SampleData[JoinedSequencesWithQuality]' \
--input-path ~/exportone/manifest.tsv \
--input-format SingleEndFastqManifestPhred33V2 \
--output-path ~/exportone/demux-joined.qza
qiime demux summarize \
--i-data ~/exportone/demux-joined.qza \
--o-visualization ~/exportone/demux-joined.qzv
qiime quality-filter q-score \
--i-demux ~/exportone/demux-joined.qza \
--p-min-quality 30 \
--p-max-ambiguous 0 \
--o-filtered-sequences ~/exportone/filtered.qza \
--o-filter-stats ~/exportone/filter-stats.qza
qiime demux summarize \
--i-data ~/exportone/filtered.qza \
--o-visualization ~/exportone/filtered.qzv
qiime deblur denoise-16S \
--i-demultiplexed-seqs ~/exportone/filtered.qza \
--p-trim-length 200 \
--p-left-trim-len 20 \
--p-sample-stats \
--p-jobs-to-start 4 \
--o-representative-sequences ~/exportone/rep-seqs-ultimate.qza \
--o-table ~/exportone/table-ultimate.qza \
--o-stats ~/exportone/deblur-stats-ultimate.qza
Primers:
- Forward (341F): 5’-CCTACGGGRSGCAGCAG-3’
- Reverse (806R): 5’-GGACTACHVGGGTWTCTAAT-3’
- Used FLASH to merge paired-end reads.
- Demultiplexed with sabre (based on barcodes) and trimmed barcode sequences.
Example sequences (from one sample):
@control1_1
GGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAACGTCAGTCATCGTCCAGAAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCTTTCCTCTCCGACACTCTAGCCTGACAGTTCCAAATGCAGTCCCGGGGTTGAGCCCCGGGCTTTCACATCTGGCTTGCCATGCCGTCTACGCTCCCTTTACACCCAGTAAATCCGGATAACGCTTGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCTTAGTCAGGTACCGTCATTATCTTCCCTGCTGATAGAGCTTTACATACCGAAATACTTCTTCGCTCACGCGGCGTCGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCCCACTGCTGCGCCCCGTAGG
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFF:FFFFFFFF:F:FFF:FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF:FFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF:FFFFFFFFF:FFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF
@control1_2
GGACTACACGGGTATCTAATCCTGTTTGATACCCACACTTTCGAGCCTCAATGTCAGTTGCAGCTTAGCAGGCTGCCATCGCAATCGGAGTTCTTAGTGATATCAAAGCATTTCAACGCTACACCACGAATTCAGCCTGCCTCAACTGCACTCAAGATATCCAGTATCAACTGCAATTTTACGGTTGAGCCGCAAACTTTCACAACTGACTTAAACATCCATCTACGCTCCCTTTAAACCCAATAAATCCGGATAACGCTCGGATCCTCGGTATTACCGCGGCGGCTGGCACGGAGTTAGCCGATCCTTATTCATAAAGTACATGCAAACGGGTATTCATACCCGACTTTATTCCTTTATAAAAGAAGTTTACAACCCATAGGGCAGTCATCCTTCACGCTACTTGGCTGGTTCAGGCTCTCGCCCATTGACCAATATTCCTCACTGCTGCGTCCC
+
FFF::FFF:FF:FF:FFFF,,:F,:,FFFFFF::FFFFFF:::F,F:,FFFF:,:F:FFF:FFFFFFF:F:FFF,,,,FFFFFFFFF:FF:,FFF:F,,FFF,F,FFF,FFFF:F,:,FFFFFF,F,F::,FF,:FFFF::F,FFF::FFF:F,FFF,F::,FFF:FFFF,F:FFF,F,FFF,:,FF,F:FFF,:FFFFFF:F:FFF:FFFFF,FFF;:,F::FFFF::FFFFFF:,FFFF:FFFF,F::F,F,,,FF:FFFFFFF:FF,F,,FF,FFFFFF:,:FFFF:FFFFF:FFF:,FFFF:FFFFFFFF:,:FFFFF,F:,::F,FFF,F,,FFFFFFF:F,FF,FFF,FF,F,::F::F,F,:FFFFFFFF:FF,F:FFF:FFFFFFFF:FFFFF::FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFF,,
@control1_3
GGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGGTTCGGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAAGTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTAACGTCAATCGTTGCGGTTATTAACCACAACGCCTTCCTCCTCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGG
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF:FFFFFFFF,,FFFF:FFFFFFFFF:FFFFFFFFFFFFFFFFF,F,FFFFFFFFFFFFF:F:FFFFFFF:FFFFFFFFFFFFF:::FFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFF:FFFFFFFFFFFFFFFFFFFFFF,,F:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF:FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFF
@control1_4
GGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCCTCAGTGTCAGTTACAGTCCAGAGAGCCGCCTTCGCAACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCACTCTCCTCTCCTGCACTCAAGTTTCCCAGTTTCAAAGGCTTACTACGGTTGAGCCGTAGCCTTTCACCTCTGACTTAAGAAACCACCTACGCACCCTTTACGCCCAGTAATTCCGGATAACGCTAGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCCTCCTCAAGTACCGTCATTATCTTCCTTGAGGACAGAGCTTTACGACCCGAAGGCCTTCATCGCTCACGCGGCGTTGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCGCC
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:F,FFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FFF:FF,FFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFF,:FFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFF:FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@control1_5
GGACTACACGGGTATCTAATCCTGTTTGCTCCCAACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATCGCCAAGGTTATTAACCTTAACGCCTTCCTCCCCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGCATCAGGCTTGCGCCAATTGTGCAATATTCCCCACTGCTGCGCCCCGTAGGTGTAATTGTCG
+
,FFF,F,FFFF,FF,FFFFFFFFFFFFF,:F:F,FF,,FFF:F:FFFFFF,F,:F:FFFFF:FFF,F,FF:,FF,F,::FFFFFF:F::F,F,FFFFFFFFFF,FFF:FFFFFFFFF:FFFF::FF:FF:FFFFFFF::FFFFFFFFF:FF,F:FFF:F:FF,F,FFFFFF,:FF,FFF,FFF:FFFFFF:FFFFF:FFFF,FF,:FFFFFFFFFFFFF:F::FF,:FFFFFFFFFF:FFFFFFFFFFFFFFFF:FFF,FF:FF,,FFFF:FFFF,:FFFF:FFF,FF,FF,,F,FFF:,:FFFFF:FFF:F:FF:FFFFFFFFFFF:FFFF:F:F,F:FFFFFF:FFFFF:FF,FFF::FFF:,,,,FFFFFFF,FF:,FFFF,:FFF:FF:,F:FFFFFFFFF:FFFFF:FFF,F::FFFFFFFFFF:,FFF::FFFFF:FF:FFF,FF,:FF:FF:FFFF,FFF:F:FFFFF,F
Any suggestions would be greatly appreciated! Why is this happening?
