metaMIC is a fully automated tool for identifying and correcting misassemblies of (meta)genomic assemblies with the following three steps. Firstly, metaMIC extracts various types of features from the alignment between paired-end sequencing reads and the assembled contigs. Secondly, the features extracted in the first step will be used as input of a random forest classifier for identifying misassembled metagenomic assemblies. Thirdly, metaMIC will localize misassembly breakpoints for each misassembled contig and then correct misassemblies by splitting into parts at the breakpoints.
Make sure you have the dependencies below installed and accessible in your $PATH.
- install python modules: pandas, numpy, pysam
conda install -c bioconda pandas numpy pysam biopython
or
pip install pysam pandas numpy biopython
- download and install samtools, bwa, jellyfish
conda install -c bioconda samtools bwa jellyfish
or
wget https://github.com/samtools/samtools/releases/download/1.9/samtools-1.9.tar.bz2
tar -jxvf samtools-1.9.tar.bz2
cd samtools-1.9
./configure
make
make install
export PATH=`pwd`:$PATH
wget https://sourceforge.net/projects/bio-bwa/files/latest/download/bwa-0.7.17.tar.bz2
tar -jxvf bwa-0.7.17.tar.bz2
cd bwa-0.7.17
make
export PATH=`pwd`:$PATH
wget http://www.cbcb.umd.edu/software/jellyfish/jellyfish-1.1.10.tar.gz
tar zxvf jellyfish-1.1.10.tar.gz
cd jellyfish-1.1.10
./configure
make
make install
export PATH=`pwd`/bin:$PATH
git clone https://github.com/ZhaoXM-Lab/metaMIC.git
- install and download training models
cd metaMIC
python setup.py install
metaMIC -h
# downloading models
metaMIC download_model
- First map paired-end reads to assembled contigs
bwa index $contig_file
bwa mem -a -t 8 $contig_file $read1 $read2 | samtools view -h -q 10 -m 50 -F 4 -b | samtools sort > $bam_file
- generate pileup file
samtools mpileup -C 50 -A -f $contig_file $bam_file | awk '$3 != "N"' > $pileup_file
- run metaMIC
# Step 1: extract features [output file: feature_matrix/window_fea_matrix.txt,feature_matrix/contig_fea_matrix.txt]
metaMIC extract_feature --bam $bam_file -c $contig_file -o $output_dir --pileup $pileup_file -m meta
# Step 2: misassembly breakpoint identification and correction;
# output directory must be same as the above $output_dir
# [output file: metaMIC_corrected_contigs.fa, misassembly_breakpoint.txt, anomaly_score.txt]
metaMIC predict -c $contig_file -o $output_dir -a MEGAHIT -m meta
# Step 1: extract features [output file: feature_matrix/window_fea_matrix.txt]
metaMIC extract_feature --bam $bam_file -c $contig_file -o $output_dir --pileup $pileup_file -m single
# Step 2: misassembly breakpoint identification and correction;
# output directory must be same as the above $output_dir
# [output file: metaMIC_corrected_contigs.fa, misassembly_breakpoint.txt, anomaly_score.txt]
metaMIC predict -c $contig_file -o $output_dir -m single
If you want to generate a new training model on a novel dataset. Contig labels and name of new training models should be provided. The Step 1 is the same as above, then the contig_fea_matrix.txt will be used as the training datasets.
# Step 1: extract features [output file: feature_matrix/window_fea_matrix.txt,feature_matrix/contig_fea_matrix.txt]
metaMIC extract_feature --bam $bam_file -c $contig_file -o $output_dir --pileup $pileup_file -m meta
# Step 2: Generating new training models
# output directory must be same as the above $output_dir
# format of contig_label file should be:
# contig1\t0
# contig2\t1
# contig3\t0
# ...
# contig100\t0
metaMIC train -o $output_dir -a $New_model_name --label $contig_label
For more details about the usage of metaMIC, read the docs
- R.sphaeroides_pileup.out
- R.sphaeroides.bam
- velvet_ctg.fasta
cd example
sh download.sh
metaMIC extract_feature --pileup R.sphaeroides_pileup.out --bam R.sphaeroides.bam -c velvet_ctg.fasta -m single -o test
metaMIC predict -c velvet_ctg.fasta -o test -m single
The output folder will contain
- Misassembly score for each contig: metaMIC_contig_score.txt (for only metagenomics)
- predicted misassembly breakpoints for misassembled contigs: misassembly_breakpoint.txt
- Anomaly scores for each position in contigs: anomaly_score.txt
- Fasta file of corrected contigs: metaMIC_corrected_contigs.fa
- Some intermediate files
For more details about the output, read the docs
metaMIC:
usage: metaMIC [-h] ...
Reference-free Misassembly Identification and Correction of metagenomic
assemblies
optional arguments:
-h, --help show this help message and exit
metaMIC subcommands:
extract_feature
Extract features from inputs.
predict Predict.
train Train model.
usage: metaMIC extract_feature [-h] [-t THREADS] [--bam BAMFILE] [--r1 READ1]
[--r2 READ2] [-p READ] -c ASSEMBLIES -o OUTPUT
--pileup PILEUP -m MODE [-l MIN_LENGTH]
[--samtools SAMTOOLS] [--jellyfish JELLYFISH]
optional arguments:
-h, --help show this help message and exit
-t THREADS, --threads THREADS
Maximum number of threads [default: 8]
--bam BAMFILE index bam file for alignment
--r1 READ1 read1
--r2 READ2 read2
-p READ, --r READ smart pairing (ignoring #2 fasta/q)
-c ASSEMBLIES, --contig ASSEMBLIES
fasta file of assembled contigs
-o OUTPUT, --output OUTPUT
output directory for metaMIC results
--pileup PILEUP path to pileup file [samtools mpileup]
-m MODE, --mode MODE Applied to single genomic/metagenomic assemblies
[meta/single]
-l MIN_LENGTH, --mlen MIN_LENGTH
Minimum contig length [default: 5000bp]
--samtools SAMTOOLS path to samtools
--jellyfish JELLYFISH
path to jellyfish
usage: metaMIC predict [-h] -o OUTPUT -m MODE -c ASSEMBLIES [-a ASSEMBLER]
[-l MIN_LENGTH] [-s SPLIT_LENGTH] [--nb BREAK_COUNT]
[--rb BREAK_RATIO] [--at ANOMALY_THRED]
optional arguments:
-h, --help show this help message and exit
-o OUTPUT, --output OUTPUT
output directory for metaMIC results
-m MODE, --mode MODE Applied to single genomic/metagenomic assemblies
[meta/single]
-c ASSEMBLIES, --contig ASSEMBLIES
fasta file of assembled contigs
-a ASSEMBLER, --assembler ASSEMBLER
The assembler-specific model or user-trained model
used for assembled fasta file [MEGAHIT/IDBA_UD/metaSPAdes/[new
training model specified by users]]
-l MIN_LENGTH, --mlen MIN_LENGTH
Minimum contig length [default: 5000bp]
-s SPLIT_LENGTH, --slen SPLIT_LENGTH
Minimum length of split fragments [default: 1000bp]
--nb BREAK_COUNT Threshold of read breakpoint counts for correcting
misassemblies
--rb BREAK_RATIO Threshold of read breakpoint ratio for correcting
misassemblies
--at ANOMALY_THRED Threshold of anomaly score for correcting
misassemblies
--st SCORE_THRED Threshold of contig score for correcting misassemblies
usage: metaMIC train [-h] -o OUTPUT [--label LABEL] [-a ASSEMBLER]
[-t THREADS]
optional arguments:
-h, --help show this help message and exit
-o OUTPUT, --output OUTPUT
output directory for metaMIC results
--label LABEL Misassembly label of contigs for training assemblies
-a ASSEMBLER, --assembler ASSEMBLER
The name of the directory of the trained model.
-t THREADS, --threads THREADS
Maximum number of CPUs [default: 8]
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