- Perl - https://www.perl.org
- Perl module "Bio::DB::Taxonomy" - https://bioperl.org
- DIAMOND - https://github.com/bbuchfink/diamond
- minimap2 - https://github.com/lh3/minimap2
- MetaPrism - https://github.com/jiwoongbio/MetaPrism
- Linux commands: sort, gzip, wget - https://www.gnu.org/software/wget/
- Centrifuge - http://www.ccb.jhu.edu/software/centrifuge/ (You can use another taxonomic identification tool.)
If you already have Git (https://git-scm.com) installed, you can get the latest development version using Git.
git clone https://github.com/jiwoongbio/NanoPrism.git
cd NanoPrism
git clone https://github.com/jiwoongbio/MetaPrism.git
Usage: perl NanoPrism_data.pl [options] [CDS.fasta [read.fastq ...]]
Options: -h display this help message
-r redownload data
-p INT number of threads [1]
-t STR target NCBI taxonomy IDs or file
-x STR minimap2 preset [map-ont]
-c FLOAT minimum coverage [0.9]
-P FILE minimap2 PAF file
-S STR stranded, "f" or "r"
-M DIR MetaPrism directory [MetaPrism]
- Create a non-redundant coding sequence database file using the given NCBI taxonomy IDs as input.
threads=8
taxonomy_id=287
cds_fasta_file=Pseudomonas_aeruginosa.CDS.fasta # output file
perl NanoPrism.pl -p $threads -t $taxonomy_id $cds_fasta_file
- Align sequencing reads to the coding sequences and determine the abundance of genes.
threads=8
taxonomy_id=287
cds_fasta_file=Pseudomonas_aeruginosa.CDS.fasta
read_fastq_file=Pseudomonas_aeruginosa.nanopore_reads.fastq.gz
perl NanoPrism.pl -p $threads -t $taxonomy_id $cds_fasta_file $read_fastq_file
- Download zymo sequencing reads
wget --no-verbose ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR315/004/ERR3152364/ERR3152364.fastq.gz
wget --no-verbose ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR315/006/ERR3152366/ERR3152366.fastq.gz
- Taxonomic classification by Centrifuge
CF_DIR=centrifuge # Centrifuge directory
CF_IDX=centrifuge/data/p_compressed # Centrifuge index filename prefix
export PATH=$CF_DIR${PATH:+:${PATH}}
sample=ERR3152364
centrifuge -p $threads -x $CF_IDX -U $sample.fastq.gz --report-file $sample.centrifuge.report.txt | gzip > $sample.centrifuge.txt.gz
sample=ERR3152366
centrifuge -p $threads -x $CF_IDX -U $sample.fastq.gz --report-file $sample.centrifuge.report.txt | gzip > $sample.centrifuge.txt.gz
- Filter 1% abundant species
sample=ERR3152364
awk -F'\t' '(NR == 1 || $3 == "species")' $sample.centrifuge.report.txt > $sample.centrifuge.report.species.txt
awk -F'\t' -va=$(awk -F'\t' '(NR > 1) {a += $6} END {print a}' $sample.centrifuge.report.species.txt) '(NR == 1 || $6 / a >= 0.01)' $sample.centrifuge.report.species.txt > $sample.centrifuge.report.species.filtered.txt
sample=ERR3152366
awk -F'\t' '(NR == 1 || $3 == "species")' $sample.centrifuge.report.txt > $sample.centrifuge.report.species.txt
awk -F'\t' -va=$(awk -F'\t' '(NR > 1) {a += $6} END {print a}' $sample.centrifuge.report.species.txt) '(NR == 1 || $6 / a >= 0.01)' $sample.centrifuge.report.species.txt > $sample.centrifuge.report.species.filtered.txt
for sample in ERR3152364 ERR3152366; do awk -F'\t' '(NR > 1) {print $2}' $sample.centrifuge.report.species.filtered.txt; done | sort -nu > species.filtered.txt
- Prepare coding sequences.
time perl NanoPrism.pl -p $threads -t species.filtered.txt NanoPrism.fasta # NanoPrism.fasta is the output file.
- Profile gene abundances
time perl NanoPrism.pl -p $threads NanoPrism.fasta ERR3152364.fastq.gz > ERR3152364.NanoPrism.abundance.txt
time perl NanoPrism.pl -p $threads NanoPrism.fasta ERR3152366.fastq.gz > ERR3152366.NanoPrism.abundance.txt
By default, the abundances are normalized by the following genes:
| Orthology | Definition |
|---|---|
| K02950 | RP-S12, MRPS12, rpsL; small subunit ribosomal protein S12 |
| K02874 | RP-L14, MRPL14, rplN; large subunit ribosomal protein L14 |
| K02946 | RP-S10, MRPS10, rpsJ; small subunit ribosomal protein S10 |
| K02948 | RP-S11, MRPS11, rpsK; small subunit ribosomal protein S11 |
| K02867 | RP-L11, MRPL11, rplK; large subunit ribosomal protein L11 |
| K02952 | RP-S13, rpsM; small subunit ribosomal protein S13 |
| K02886 | RP-L2, MRPL2, rplB; large subunit ribosomal protein L2 |
| K02988 | RP-S5, MRPS5, rpsE; small subunit ribosomal protein S5 |
| K02992 | RP-S7, MRPS7, rpsG; small subunit ribosomal protein S7 |
| K02965 | RP-S19, rpsS; small subunit ribosomal protein S19 |
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