You can download Recycler here or clone it via the link below. In case you download the zip, unzip the file before following the instructions below (ignoring the 'git clone' line)
To install Recycler and scripts follow the following instructions.
git clone https://github.com/Shamir-Lab/Recycler.git
cd Recycler
python setup.py install --user
Assuming we have prepared a filtered BAM file (aln-pe.bam) prepared as described below and an isolate assembly graph (e.g., assembly_graph.fastg from SPAdes 3.6+), and that 55 was the maximum k-mer length used by the assembler,
recycle.py -g assembly_graph.fastg -k 55 -b aln-pe.bam -i True
For metagenome/plasmidome assemblies, we remove the final ("-i") parameter, which has a default False value.
Recycler is a tool designed for extracting circular sequences from de novo assembly graphs. It can be applied on isolate as well as metagenome and plasmidome data. The circular sequences it outputs may be plasmids, phages, etc. Recycler uses only features of the assembly graph (contig overlaps, lengths, and coverage level information) and alignments of paired-end reads to the contigs in the graph in order to identify these sequences.
Recycler is implemented in Python, and has been tested only on Python 2.7+. We recommend using the Anaconda distribution to ease package installations. Recycler requires the following packages be installed:
Recommended for generating inputs (as used during testing):
recycle.py -g GRAPH -k MAX_K -b BAM [-l LENGTH] [-m MAX_CV] [-i ISO] [-o OUTPUT_DIR]
-g GRAPH
(spades 3.50+) assembly graph FASTG file to process:
for spades 3.5, before_rr.fastg; for spades 3.6+, assembly_graph.fastg
-k MAX_K
integer reflecting maximum k value used by the assembler
-b BAM
BAM file resulting from aligning reads to contigs file, filtering for best matches
-l LENGTH
minimum length required for reporting [default: 1000]
-m MAX_CV
coefficient of variation used for pre-selection
[default: 0.5, higher--> less restrictive]
-i ISO
True or False value reflecting whether data sequenced
was an isolated strain
-o OUTPUT_DIR
provide a specific output directory by default results will
be written to the directory the FASTG file is currently in.
Recycler uses paired-end alignments of the reads originally assembled to the output assembly graph to filter and select amongst candidate circular sequences. In order to do so, it requires as input a BAM file containing the set of best alignment hits for each read pair. We recommend the following steps (tested on BWA 0.7.5 and samtools 1.19) to prepare the BAM file:
make_fasta_from_fastg.py -g assembly_graph.fastg [-o assembly_graph.nodes.fasta]
bwa index assembly_graph.nodes.fasta
bwa mem assembly_graph.nodes.fasta R1.fastq.gz R2.fastq.gz | samtools view -buS - > reads_pe.bam
samtools view -bF 0x0800 reads_pe.bam > reads_pe_primary.bam
samtools sort reads_pe_primary.bam reads_pe_primary.sort.bam
samtools index reads_pe_primary.sort.bam
following these steps, we only need the files reads_pe_primary.sort.bam and reads_pe_primary.sort.bam.bai.
- <prefix>.cycs.fasta - a fasta formatted file of predicted plasmids
- <prefix>.cycs.paths_w_cov.txt - a text file containing information about plasmids composed of multiple contigs.
The format for the second file is:
- <plasmid name> - e.g., RNODE_5_length_42666_cov_19.93685
- <node names in the original graph making up this cycle> - e.g., ('NODE_2801_length_42596_cov_19.8677', "NODE_2387_length_125_cov_34.7286'").
- <coverage levels of nodes at the time they are removed> - e.g., [19.8677, 34.7286]
- <node numbers in the original graph making up this cycle> - e.g., [2801, 2387]. This can be useful for visualizing the path in tools like Bandage
We are aware other tools can be used to generate the inputs to Recycler - e.g., Bowtie2 for BAM files and Megahit for FASTGs, - however, we have not tested them with Recycler. We welcome user feedback regarding this point, and compatibilty issues that arise may be posted to the issues tracker above!
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