flopp is a software package for single individual haplotype phasing of polyploid organisms from long read sequencing. flopp is extremely fast, multithreaded, and written entirely in the rust programming language. flopp offers an order of magnitude speedup and better accuracies compared to other polyploid haplotype phasing algorithms.
Given
- a list of variants in .vcf format
- a set of reads mapped to a reference in .bam format
for a single individual, flopp outputs a set of phased haplotypes.
- rust and associated tools such as cargo are required and assumed to be in PATH.
flopp is installable as a bioconda package using
conda install -c bioconda floppor if compiling through Rust
git clone https://github.com/bluenote-1577/flopp
cd flopp
cargo build --release
./target/release/flopp -hcargo build --release builds the flopp binary, which is found in the ./target/release/ directory.
# with VCF + BAM. "-v" constrains phasing by VCF genotypes.
flopp -b bamfile.bam -v vcffile.vcf -p (ploidy) -o results.txt -P partition_directory
# with VCF + BAM. "-c" ignores genotyping information in VCF e.g. diploid or population VCFs.
# Use this if your VCF and reads come from different samples.
flopp -b bamfile.bam -c vcffile.vcf -p (ploidy) -o unpolished_results.txt -P partition_directory
# with fragment file
flopp -f fragfile.frags -p (ploidy) -o unpolished_results.txt The ploidy of the organism must be specified. The number of threads (default 10) can be specified using the -t option. See flopp -h for more information.
For a quick test, we provide a VCF and BAM files in the tests folder. Run
# clone this repo if you haven't
git clone https://github.com/bluenote-1577/flopp
cd flopp
# assuming flopp is in PATH
flopp -b tests/test_bams/pds_ploidy3.bam -v tests/test_vcfs/pds.vcf -p 3 -o results.txt -P test_partition_directoryto run flopp on a 3 Mb section of a simulated 3x ploidy potato chromosome with 30x read coverage.
The standard mode of usage is to specify a bam file using the option -b and a vcf file using the option -v or -c.
- Use -v to constrain the phasing by the the genotypes in your .vcf file. Only works if the ploidy is in the vcf is the same as the -p option.
- Use -c to ignore the genotyping information in the vcf. If the .vcf file is generated from a different sample, the ploidy is not the same as the -p option, or if you're not confident in the genotyping calls then use this option.
The output is written to a text file with value of option -o. If -P is specified, then the partition of the input reads according to haplotypes is also output.
flopp currently only uses SNP information and does not take into account indels. However, the user may define their own fragments which can be indexed by other types of variants. See the Fragment file section at the bottom.
The bam file may contain multiple contigs/references which the reads are mapped to as long as the corresponding contigs also appear in the vcf file.
flopp outputs a phased haplotype file in the following format:
**contig name** k columns k columns
(snp #1):(genome position) (inferred allele #: 0/1/2...) ... (allele #1):(support)|(allele #2):(support)|...
(snp #2):(genome position) (inferred allele #: 0/1/2...) ... (allele #1):(support)|(allele #2):(support)|...
...
- Column 1 is (variant) : (genome position) where (variant) is the i-th variant, and (genome position) is the the position of the variant on the reference.
- The next k columns are the k phased haplotypes for an organism of ploidy k. 0 represents the reference allele, 1 the first alternate, and so forth.
- The next k columns are of the form (allele):(support)|(allele):(support) where (allele) = 0,1,... and (support) is the number of reads assigned to the specific haplotype for that allele. For example, 0:10|1:5 indicates that 10 reads assigned to this haplotype have allele 0 at this position, and 5 reads have allele 1.
If at an allele, no reads corresponding to a haplotype cover the allele, -1 is output as the consensus and NA is written in the last column.
If using a bam file with multiple contigs being mapped to, the output file contains multiple phased haplotypes of the above format which are delimited by **(contig name)**.
If also using -P option, flopp outputs the read partition obtained by flopp. That is, set of reads corresponding to each haplotype. The format looks like:
#1 (partition #1)
(read_name1) (first SNP position covered) (last SNP position covered)
(read_name2) (first SNP position covered) (last SNP position covered)
...
#2 (partition #2)
...
To create a new BAM with the HP:i:x tags where x is 0,1,2,... indicates which haplotype the read belongs to, use
python scripts/haplotag_bam.py contig_part.txt original_input_bam.bam new_haplotagged_bam_name.bam
Where contig_part.txt is a file in the directory specified by the -P option. This script requires pysam to be installed. This only works for one contig at a time for now.
To get a set of BAM files which correspond to the output read partition (i.e. the haplotypes), use
python scripts/get_bam_partition.py contig_part.txt original_input_bam.bam prefix_name
This will output a set of bams labelled prefix_name1.bam, prefix_name2.bam and so forth. This script requires pysam.
A user can also input a fragment file using the option -f. The fragment file is a file where each line is a read which is indexed by variants; see https://github.com/MinzhuXie/H-PoPG or https://github.com/realabolfazl/AltHap (but be careful, see issue) for more details about the fragment file specifcation (called the input snp matrix by H-PoP). Specifying a compatible VCF file with a fragment file uses genotyping information to produce a higher quality output; only SNPs will be processed in the VCF.
For testing purposes and compatibility with other haplotype phasing algorithms, the binary frag-dump is provided in the same folder as the flopp binary.
frag-dump -b bamfile.bam -v vcffile.vcf -o frags.txt gives a fragment file a.k.a input snp matrix which is compatible with H-PoP and other haplotype phasing algorithms.
We found that some variant callers don't put contig headers in the VCF file. In this situation, run python scripts/write_contig_headers_vcf.py (vcf_file) to get a new VCF with contig headers.
- Fixed some 0-index out of bounds bugs caused by secondary alignments and very short read fragments
- Fixed a bug where supplementary alignments were used incorrectly. Only primary alignments are used in the BAM file now.
- Added options; user can now manipulate error rates, block lengths, and more
- flopp can now output the read partition obtained by phasing using the -P option
- flopp outputs MEC to stdout by default now
- fixed a bug where the UPEM normalization was too small
- Can now input non-polyploid VCF file. If your genotypes are not confident or if you use a diploid variant caller, you can use -c (VCF) instead.
Jim Shaw and Yun William Yu. flopp: Extremely Fast Long-Read Polyploid Haplotype Phasing by Uniform Tree Partitioning (2022). Journal of Computational Biology: RECOMB 2021 Issue.