MGSE can harness the power of files generated in genome sequencing projects to predict the genome size. Required are the FASTA file containing a high continuity assembly and a BAM file with all available reads mapped to this assembly. The script construct_cov_file.py (https://doi.org/10.1186/s12864-018-5360-z) allows the generation of a COV file based on the (sorted) BAM file (also possible via MGSE directly). Next, this COV file can be used by MGSE to calculate the coverage in provided reference regions and to calculate the total number of mapped bases. Both values are subjected to the genome size estimation. Providing accurate reference regions is crucial for this genome size estimation. Different alternatives were evaluated and actual single copy BUSCOs (https://busco.ezlab.org/) appear to be the best choice. Running BUSCO prior to MGSE will generate all necessary files.
Usage:
python3 MGSE3.py [--cov <COV_FILE_OR_DIR> | --bam <BAM_FILE_OR_DIR> | --fasta <FASTA_FILE>
--fastq <FASTQ_FILE_OR_DIR>]
--out <DIR>
[--ref <TSV> | --gff <GFF> | --busco <FULL_TABLE.TSV> | --all]
Mandatory:
Coverage data (choose one)
[--cov STR Coverage file (COV) created by construct_cov_file.py or directory containing
multiple coverage files]
[--bam STR BAM file to automatically create the coverage file]
[--fasta STR FASTA assembly file for read mapping
--fastq STR Single FASTQ file or a directory containing FASTQ files]
Output directory
--out STR Output directory
Reference regions to calculate average coverage (choose one)
--ref STR TAB-separated file with sequence ID, start position, and end position
--gff STR GFF3 file
--busco STR BUSCO annotation file (full_table.tsv)
--all Use all positions of the assembly
Optional:
--black STR Sequence ID list for exclusion
--samtools STR Full path to samtools (if not in your $PATH)
--bedtools STR Full path to bedtools (if not in your $PATH)
--seqtech STR Sequencing technology of FASTQ (illumina|ont|pacbio)[illumina]
--short_read_aligner STR Full path to BWA MEM (if not in your $PATH)
--long_read_aligner STR Full path to minimap2 (if not in your $PATH)
--name STR Prefix for output files []
--feature STR Specific feature for analysis from GFF file (if other than 'gene')
--m INT Samtools sort memory [5000000000]
--threads INT Samtools sort threads [4]
--plot BOOLEAN Activate or deactivate generation of figures via matplotlib[FALSE]
--black_list_factor FLOAT Black list factor for blacklisting of contigs with high coverage values [1.5]
--blackoff BOOLEAN Deactivate the black listing of contigs with high coverage values [FALSE]
--gzip Search for files "*cov.gz" in --cov if this is a directory
--gzip_fq Specify this flag if the FASTQ file(s) are compressed
--bam_is_sorted Do not sort BAM file
WARNING:
- MGSE requires absolute paths (at least use of absolute paths is recommended)
- Per default contigs with very high coverage values are put on a black list to prevent inflation of the genome size prediction by plastome contigs (in plants). However, this function can be disabled via --blackoff to estimate genome sizes with more fragmented assemblies.
Possible reference regions:
-
--refA very simple TAB-separated text file with information about chromosome, start, and end of regions which should be used as a reference set for the coverage calculation. -
--gffA GFF3 file with genes which should serve as reference regions. -
--buscoThis will extract the single copy BUSCOs from the provided TSV file. -
--allAll positions of the assembly will be included in the average coverage calculation.
Important pointers:
-
'--fastq' If you have multiple FASTQ files, It is mandatory to put them in a folder and specify the folder path. While specifying the folder path ensure that it ends with a backslash '/' since it is mandatory for the script to work correctly.
-
'--cov' Extension of uncompressed cov file should be .cov or .txt; Compressed cov file extension should be .cov.gz or.txt.gz
Usage:
python2 construct_cov_file.py
--in <BAM_FILE> --out <OUTPUT_FILE>
Mandatory:
--in STR Bam file
--out STR Output file
Optional:
--bam_is_sorted Do not sort BAM file
--m INT Samtools sort memory [5000000000]
--threads INT Samtools sort threads [4]
--samtools STR Full path to samtools (if not in your $PATH)
--bedtools STR Full path to bedtools (if not in your $PATH)
Natarajan S., Gehrke J., Pucker B. (2024). Mapping-based genome size estimation. bioRxiv 607390; doi: 10.1101/607390.