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fix bugs from the example dataset
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@Hydro3639
Hydro3639 authored Oct 17, 2023
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## A guide to verify NanoPhase installation: reconstructing reference-quality MAGs from the ZymoBIOMICS dataset (nanophase v0.2.3)
### ZymoBIOMICS dataset Download
Note: this dataset is generated from the [ZymoBIOMICS gut microbiome standard](https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-022-01415-8#:~:text=The%20ZymoBIOMICS%20gut,microbiome%2Dstandard.) and used for feasibility evaluation in our [manuscript](https://doi.org/10.1186/s40168-022-01415-8)); in addition, `fastq-dump` in [SRA toolkit](https://github.com/ncbi/sra-tools/wiki/01.-Downloading-SRA-Toolkit) is required.
```
fastq-dump SRR17913199
```

### Using only Nanopore long reads to reconstruct reference-quality MAGs
```
conda activate nanophase ## if not in the nanophase env
nanophase meta -l SRR17913199.fastq -t 64 -o nanophase-out ## using only Nanopore long reads
```
It may take a while to finish. The output folder/file structure should be like:
```
nanophase-out/
├── 01-LongAssemblies
│ ├── assembly_graph.gfa
│ ├── assembly.fasta
│ ├── assembly_info.txt
│ └── flye.log
├── 02-LongBins
│ ├── INITIAL_BINNING
│ │ ├── metabat2
│ │ ├── tmp.abun.txt
│ │ ├── maxbin2
│ │ └── semibin
│ └── BIN_REFINEMENT
│ ├── maxbin2-bins
│ ├── maxbin2-bins.stats
│ ├── metabat2-bins
│ ├── metabat2-bins.stats
│ ├── metawrap_50_10_bins
│ ├── metawrap_50_10_bins.stats
│ ├── semibin-bins
│ ├── semibin-bins.stats
│ ├── figures
│ ├── bin_refinement.log
│ └── metawrap_50_10_bins.contigs
└── 03-Polishing
├── Racon
│ ├── racon.polish.log
│ ├── bin.1
│ ├── bin.10
│ ├── bin.11
│ ├── bin.12
│ ├── bin.2
│ ├── bin.3
│ ├── bin.4
│ ├── bin.5
│ ├── bin.6
│ ├── bin.7
│ ├── bin.8
│ └── bin.9
├── medaka
│ ├── bin.4-medaka
│ ├── bin.10-medaka
│ ├── bin.3-medaka
│ ├── bin.7-medaka
│ ├── bin.1-medaka
│ ├── bin.12-medaka
│ ├── bin.6-medaka
│ ├── bin.2-medaka
│ ├── bin.8-medaka
│ ├── bin.5-medaka
│ ├── bin.11-medaka
│ ├── bin.9-medaka
│ └── medaka.polish.log
├── checkm-result
│ ├── bins
│ ├── lineage.ms
│ ├── checkm.log
│ ├── checkm_results.tsv
│ └── storage
├── gtdbtk-result
│ ├── gtdbtk.warnings.log
│ ├── gtdbtk.ar53.summary.tsv -> classify/gtdbtk.ar53.summary.tsv
│ ├── align
│ ├── classify
│ ├── gtdbtk.bac120.summary.tsv -> classify/gtdbtk.bac120.summary.tsv
│ ├── gtdbtk.json
│ ├── gtdbtk.log
│ └── identify
├── nanophase.ont.genome.summary
└── Final-bins
├── bin.10.fasta
├── bin.11.fasta
├── bin.12.fasta
├── bin.1.fasta
├── bin.2.fasta
├── bin.3.fasta
├── bin.4.fasta
├── bin.5.fasta
├── bin.6.fasta
├── bin.7.fasta
├── bin.8.fasta
└── bin.9.fasta
48 directories, 34 files
```
Final reconstructed MAGs could be found in `nanophase-out/03-Polishing/Final-bins/` and the summary file of the reconstructed MAGs could be found in `nanophase-out/03-Polishing/nanophase.ont.genome.summary`
```
ls nanophase-out/03-Polishing/Final-bins/
bin.10.fasta bin.11.fasta bin.12.fasta bin.1.fasta bin.2.fasta bin.3.fasta bin.4.fasta bin.5.fasta bin.6.fasta bin.7.fasta bin.8.fasta bin.9.fasta
cat nanophase-out/03-Polishing/nanophase.ont.genome.summary
#BinID Completeness Contamination Strain heterogeneity GenomeSize(bp) N_Contig N50(bp) GC GTDB-Taxa
bin.1 100.00 0.00 0.00 2461381 2 2150631 0.26980 d__Bacteria;p__Fusobacteriota;c__Fusobacteriia;o__Fusobacteriales;f__Fusobacteriaceae;g__Fusobacterium;s__Fusobacterium animalis
bin.2 99.44 0.29 0.00 3460839 1 3460839 0.48731 d__Bacteria;p__Bacillota_A;c__Clostridia;o__Lachnospirales;f__Lachnospiraceae;g__Roseburia;s__Roseburia hominis
bin.3 99.18 0.55 100.00 1906542 3 1890918 0.52286 d__Bacteria;p__Bacillota;c__Bacilli;o__Lactobacillales;f__Lactobacillaceae;g__Limosilactobacillus;s__Limosilactobacillus fermentum
bin.4 95.88 0.00 0.00 2849782 2 2341158 0.55459 d__Bacteria;p__Verrucomicrobiota;c__Verrucomicrobiae;o__Verrucomicrobiales;f__Akkermansiaceae;g__Akkermansia;s__Akkermansia muciniphila
bin.5 100.00 0.00 0.00 2157911 1 2157911 0.38952 d__Bacteria;p__Bacillota_C;c__Negativicutes;o__Veillonellales;f__Veillonellaceae;g__Veillonella;s__Veillonella rogosae
bin.6 87.91 0.39 0.00 2522000 3 2083939 0.43694 d__Bacteria;p__Bacteroidota;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Prevotella;s__Prevotella corporis
bin.7 99.16 0.00 0.00 2028285 1 2028285 0.59292 d__Bacteria;p__Actinomycetota;c__Actinomycetia;o__Actinomycetales;f__Bifidobacteriaceae;g__Bifidobacterium;s__Bifidobacterium adolescentis
bin.8 100.00 0.00 0.00 2911148 1 2911148 0.57806 d__Bacteria;p__Bacillota_A;c__Clostridia;o__Oscillospirales;f__Ruminococcaceae;g__Faecalibacterium;s__Faecalibacterium prausnitzii_E
bin.9 95.94 0.50 0.00 5191902 5 4733348 0.43263 d__Bacteria;p__Bacteroidota;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides;s__Bacteroides fragilis
bin.10 81.03 0.00 0.00 4065942 1 4065942 0.28625 d__Bacteria;p__Bacillota_A;c__Clostridia;o__Peptostreptococcales;f__Peptostreptococcaceae;g__Clostridioides;s__Clostridioides difficile
bin.11 98.03 0.97 80.00 4491398 26 302533 0.50954 d__Bacteria;p__Pseudomonadota;c__Gammaproteobacteria;o__Enterobacterales;f__Enterobacteriaceae;g__Escherichia;s__Escherichia coli
bin.12 58.04 0.00 0.00 1191563 44 34294 0.30992 d__Archaea;p__Methanobacteriota;c__Methanobacteria;o__Methanobacteriales;f__Methanobacteriaceae;g__Methanobrevibacter_A;s__Methanobrevibacter_A smithii
```
### If you had Illumina short reads as well, you may use hybrid strategy (Nanopore long reads + Illumina short reads) to reconstruct reference-quality MAGs
Note: since nanopore sequencing has been improved quite a lot, at this time being, short reads is not necessary; especially for long data generated from the Kit 14.
```
conda activate nanophase ## if not in the nanophase env
nanophase meta -l lr.fa.gz --hybrid -1 sr_1.fa.gz -2 sr_2.fa.gz -t 40 -o hybrid-nanophase-out ## using Nanopore + Illumina reads
```


# The following tutorial is suitable for nanophase v0.2.2 due to the limited dataset provided
## A guide to verify NanoPhase installation: reconstructing reference-quality MAGs from an example dataset (nanophase v0.2.2)
#### Warning: since nanophase v0.2.3 ([semibin](https://github.com/BigDataBiology/SemiBin) as a new binner, which really improved genome reconstruction results a lot for complex environmental samples), it may encounter error issues when performing binning process with only a few contigs. It should also be an issue when handling very simple long-read datasets, so in this way, I would suggest to use nanophase v0.2.2: `mamba create -n nanophase-v0.2.2 -c nanophase nanophase=0.2.2 -y`. Sorry for that, we will try to fix this issue in the next release.
#### Warning: since nanophase v0.2.3 ([semibin](https://github.com/BigDataBiology/SemiBin) as a new binner, which really improved genome reconstruction results a lot for complex environmental samples), it may encounter error issues when performing the binning process with only a few contigs. It should also be an issue when handling very simple long-read datasets, so in this way, I would suggest using nanophase v0.2.2: `mamba create -n nanophase-v0.2.2 -c nanophase nanophase=0.2.2 -y`. Sorry for that, we will try to fix this issue in the next release.
### [Example dataset](https://github.com/example-data/np-example) download
```
wget https://github.com/example-data/np-example/raw/main/np.test.tar && tar -xvf np.test.tar && rm -rf np.test.tar ## download the example data
```
Or it waits too long, you may using the following link as well
Or it waits too long, you may use the following link as well
```
wget -O np.test.tar https://www.dropbox.com/s/ey9hfntqn789gz0/np.test.tar?dl=0 && tar -xvf np.test.tar && rm -rf np.test.tar
```
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