The easiest way to install Liftoff is with the conda package manager.
conda install -c bioconda liftoff
If you don't have conda installed, you need to install Minimap2 (following instructions here) and Liftoff from source or with pip.
git clone https://github.com/agshumate/Liftoff liftoff
cd liftoff
python setup.py install
pip install Liftoff
usage: liftoff [-h] (-g GFF | -db DB) [-o FILE] [-u FILE] [-exclude_partial]
[-dir DIR] [-mm2_options =STR] [-a A] [-s S] [-d D] [-flank F]
[-V] [-p P] [-m PATH] [-f TYPES] [-infer_genes]
[-infer_transcripts] [-chroms TXT] [-unplaced TXT] [-copies]
[-sc SC] [-overlap O] [-mismatch M] [-gap_open GO]
[-gap_extend GE]
target reference
Lift features from one genome assembly to another
Required input (sequences):
target target fasta genome to lift genes to
reference reference fasta genome to lift genes from
Required input (annotation):
-g GFF annotation file to lift over in GFF or GTF format
-db DB name of feature database; if not specified, the -g
argument must be provided and a database will be built
automatically
Output:
-o FILE write output to FILE in GFF3 format; by default, output
is written to terminal (stdout)
-u FILE write unmapped features to FILE; default is
"unmapped_features.txt"
-exclude_partial write partial mappings below -s and -a threshold to
unmapped_features.txt; if true partial/low sequence
identity mappings will be included in the gff file with
partial_mapping=True, low_identity=True in comments
-dir DIR name of directory to save intermediate fasta and SAM
files; default is "intermediate_files"
Alignments:
-mm2_options =STR space delimited minimap2 parameters. By default ="-a
--end-bonus 5 --eqx -N 50 -p 0.5"
-a A designate a feature mapped only if it aligns with
coverage ≥A; by default A=0.5
-s S designate a feature mapped only if its child features
(usually exons/CDS) align with sequence identity ≥S; by
default S=0.5
-d D distance scaling factor; alignment nodes separated by
more than a factor of D in the target genome will not be
connected in the graph; by default D=2.0
-flank F amount of flanking sequence to align as a fraction
[0.0-1.0] of gene length. This can improve gene
alignment where gene structure differs between target
and reference; by default F=0.0
Miscellaneous settings:
-h, --help show this help message and exit
-V, --version show program version
-p P use p parallel processes to accelerate alignment; by
default p=1
-m PATH Minimap2 path
-f TYPES list of feature types to lift over
-infer_genes use if annotation file only includes transcripts,
exon/CDS features
-infer_transcripts use if annotation file only includes exon/CDS features
and does not include transcripts/mRNA
-chroms TXT comma seperated file with corresponding chromosomes in
the reference,target sequences
-unplaced TXT text file with name(s) of unplaced sequences to map
genes from after genes from chromosomes in chroms.txt
are mapped; default is "unplaced_seq_names.txt"
-copies look for extra gene copies in the target genome
-sc SC with -copies, minimum sequence identity in exons/CDS for
which a gene is considered a copy; must be greater than
-s; default is 1.0
-overlap O maximum fraction [0.0-1.0] of overlap allowed by 2
features; by default O=0.1
-mismatch M mismatch penalty in exons when finding best mapping; by
default M=2
-gap_open GO gap open penalty in exons when finding best mapping; by
default GO=2
-gap_extend GE gap extend penalty in exons when finding best mapping;
by default GE=1
The only required inputs are the reference genome sequence(fasta format), the target genome sequence(fasta format) and the reference annotation or feature database. If an annotation file is provided with the -g argument, a feature database will be built automatically and can be used for future lift overs by providing the -db argument. All files, i.e. gff3 and fasta, must be present in writable directories as indices will be created in place by the tool.
By default, 'gene' features and all child features of genes (i.e. trancripts, mRNA, exons, CDS, UTRs) will be lifted over. The -f parameter can be used to specify a file containing a list of additional parent feature types you wish to lift-over. Note: feature IDs must be unique for every feature and may not contain spaces. Example of a feature types file would be the following:
biological_region
miRNA
repeat_element
A gene will be considered mapped successfully if the alignment coverage and sequence identity in the child features (usually exons/CDS) is >= 50%. This can be changed with the -a and -s options. By default, genes that map below these thresholds will be included in the gff file with partial_mapping=True and low_identity=True in the last column. To exclude these partial/low identity mappings from the final GFF use -exclude_partial, and these genes will instead be written to the unmapped_features.txt file. The sequence identity and alignment coverage is reported in the final column of the output GFF for feach gene.
By default liftoff uses the following parameters for the minimap2 alignments -a --eqx --end-bonus 5 -N 50 -p 0.5
-a and --eqx specify that the output should be in SAM format with the cigar string including "=" for matches and "X" for mismatches (opposed to the default SAM format using 'M' for both). The -N and -p parameters specficied allow for more secondary alignments to be considered which is helpful in the resolution of multi-gene families. The --end-bonus parameter favors end-to-end alignments of the gene over soft clipping a mismatched base at the start or end of the alignment. For example if the stop codon of the reference gene is TAA and the stop codon of the target gene is TAG, without the end-bonus parameter, this alignment and subsequent annotation would be truncated by 1 base.
The user may wish to change the minimap2 parameters for their specific data. This can be done with the -mm2_options parameter with a string of options to add/change preceeded by an "=" sign. The "=" is important as it distinguishes minimap2 parameters from liftoff parameters with the same flag. For more divergent species in particular, increasing the -r and -z parameters may improve results (see Minimap2 documentation for more details). An example of changing these with -mm2_options would be
-mm2_options="-r 2k -z 5000"
With the -polish option Liftoff will re-align the exons in attempt to restore proper coding sequences in cases where the lift-over resulted in start/stop codon loss or introduced an in-frame stop codon. This will increase the run time but offers improvments in preserving proper CDS annotations. With the polish option, 2 output GFF/GTF files will be created named {output}.gff and {output}.gff_polished. {output}.gff contains the annotations prior to the polishing step and {output}.gff_polished contains the annotations after being polished.
Liftoff works best when the gene structure (i.e intron size) is similar in the reference and target genomes. When genes differ significantly in size, the alignments are more fragmented and often small exons at the beginning or end of the gene are not aligned. Adding and aligning some percentage of flanking sequence to the gene with the -flank option can improve this in some cases. Additionally increasing the -d parameter will allow mappings where the genes are much larger in the target genome than in the reference.
By default, all genes will be aligned to the entire target assembly. However, for chromosome-scale assemblies of the same species, the -chroms option can be used to perform the lift-over chromosome by chromosome which improves accuracy. After the chromosome by chromosome lift over is complete, any genes that did not map will be aligned to the whole genome. This is strongly recommended for repetitive/polyploid genomes where there are many similar genes on different chromosomes. This option can be enabled by providing a comma separated file chroms.txt with corresponding chromosome names with the -chroms argument. Each line of the file should follow {ref_chrom_name},{target_chrom_name} for each pair of corresponding chromosomes. For example, a lift over from a Genbank human assembly to a Refseq human assembly would have the following chroms.txt file.
chr1,NC_000001.10
chr2,NC_000002.11
chr3,NC_000003.11
chr4,NC_000004.11
chr5,NC_000005.9
chr6,NC_000006.11
chr7,NC_000007.13
chr8,NC_000008.10
chr9,NC_000009.11
chr10,NC_000010.10
chr11,NC_000011.9
chr12,NC_000012.11
chr13,NC_000013.10
chr14,NC_000014.8
chr15,NC_000015.9
chr16,NC_000016.9
chr17,NC_000017.10
chr18,NC_000018.9
chr19,NC_000019.9
chr20,NC_000020.10
chr21,NC_000021.8
chr22,NC_000022.10
chrX,NC_000023.10
chrY,NC_000024.9
A list of unplaced sequence names can be provided with the -unplaced option. With this option, genes from these unplaced contigs in the reference will be mapped to the target assembly after the genes on the main chromosomes in the chroms.txt have been mapped.
With the -copies option, Liftoff will look for extra copies of genes that are not annotated in the reference after the initial lift over. A gene copy will only be annonated at a locus if it does not overlap another annotated feature. By default, exons/CDS's must have 100% sequence identity Extra gene copies will have the same ID as the reference gene and will be tagged with extra_copy_number={copy_number} in the last column of the GFF file.
The output is a file in the same format as the reference annotation (GFF3 or GTF) for the target genome and a file with the IDs of unmapped genes. The 9th column of the target annotation will contain the same information as the original reference plus the following
sequence_ID: The sequence identity of the gene compared to the reference in exon regions
coverage: The alignment coverage of the gene in exon regions
valid_ORFs: The number of valid ORFs annotated within the gene
valid_ORF: Indicates the CDS annotation properly starts with a start codon, ends with a stop codon,
and does not have any in-frame stop codons.
matches_ref_protein: Indicates the translated CDS matches the reference CDS exactly
missing_start_codon: Indicates the CDS does not begin with a start codon
missing_stop_codon: Indicates the CDS does not end with a stop codon
inframe_stop_codon: Indicates the CDS has an inframe stop codon.
extra_copy_number: The copy number increase of this feature compared to the reference.
extra_copy_number=0 means this is the original reference gene.
Extracting gene sequences from bgzipped files is possible but much slower. It is recommended to decompress bgzipped FASTA files first.
If you use Liftoff in your work please cite
Shumate, Alaina, and Steven L. Salzberg. 2020. “Liftoff: Accurate Mapping of Gene Annotations.” Bioinformatics , December. https://doi.org/10.1093/bioinformatics/btaa1016.