EviAnn (Evidence Annotation) is novel genome annotation software. It is purely evidence-based. EviAnn derives protein-coding gene and long non-coding RNA annotations from RNA-seq data and/or transcripts, and alignments of proteins from related species. EviAnn outputs annotations in GFF3 format. EviAnn does not require genome repeats to be soft-masked prior to running annotation. EviAnn is stable and fast. Annotation of a mouse (M.musculus) genome takes less than one hour on a single 24 core Intel Xeon Gold server (assuming input of aligned RNA-seq reads in BAM format and ~346Mb of protein sequences from several related species including human).
Benefits of using EviAnn:
- EviAnn's output is fully compliant with NCBI annotation specifications, annotations can be easily submitted to NCBI GenBank using table2asn tool (see below)
- Easy to install and run, few easy to install dependncies
- Eviann is very fast -- annotation of a mammalian genome takes less than an hour, after all RNA-seq data has been aligned
- 5' and 3' UTR annotations for most protein-coding transcripts
- Annotates long non-coding RNA's
- Processed pseudo-genes are automatically labelled and CDSs for them are not reported
- Optional automatic functional annotation with UniProt-SwillProt database (-f switch)
- Support for long and short transcriptome sequencing reads and mixed data sets
- Support for genomes up to 32Gbp in size
- If one or more close relatives are annotated, annotation is possible with transcripts and proteins from the related genomes, without any RNA-seq data. Genomes must be >95% similar on the DNA level.
Development of EviAnn is supported in part by NSF grant IOS-2432298, and by NIH grants R01-HG006677 and R35-GM130151.
To install, first download the latest distribution tarball EviAnn-X.X.X.tar.gz (not one of the Source code files!) from the github release page https://github.com/alekseyzimin/EviAnn_release/releases. Replace X's below with the version number. Then run:
$ tar xvzf EviAnn-X.X.X.tar.gz
$ cd EviAnn-X.X.X
$ ./install.sh
The installation script will configure and make all necessary packages. The EviAnn executables will appear under bin/. You can run EviAnn from anywhere by executing /path_to/EviAnn-X.X.X/bin/eviann.sh
EviAnn requires the following external dependencies to be installed and available on the $PATH:
- minimap2: https://github.com/lh3/minimap2
- HISAT2: https://github.com/DaehwanKimLab/hisat2
Here is the list of the dependencies included with the package:
- StringTie version 2.2.1 -- static executable
- gffread version 0.12.7 -- static executable
- gffread version 0.12.6 -- static executable
- blastp version 2.13.0+ -- static executable
- tblastn version 2.13.0 -- static executable
- makeblastdb version 2.13.0 -- static executable
- exonerate version 2.4.0 -- static executable
- TransDecoder version 5.7.1
- samtools version 0.1.20
- ufasta version 1
- miniprot v0.13 -- static executable
You can clone the development tree, but then there are dependencies such as swig and yaggo (http://www.swig.org/ and https://github.com/gmarcais/yaggo) that must be available on the PATH:
$ git clone https://github.com/alekseyzimin/EviAnn_release
$ cd EviAnn_release
$ git submodule init
$ git submodule update
$ cd ../ufasta && git checkout master
$ cd ..
$ make
$ (cd build/inst/bin && tar xzf TransDecoder-v5.7.1.tar.gz)
To create a distribution, run 'make install'. Run 'make' to compile the package. The binaries will appear under build/inst/bin. The name of the distribution package is specified at the top of the Makefile. Note that on some systems you may encounter a build error due to lack of xlocale.h file, because it was removed in glibc 2.26. xlocale.h is used in Perl extension modules used by EviAnn. To work around this error, you can upgrade the Perl extensions, or create a symlink for xlocale.h to /etc/local.h or /usr/include/locale.h, e.g.:
ln -s /usr/include/locale.h /usr/include/xlocale.h
Usage: eviann.sh [options]
Options:
-t INT number of threads, default: 1
-g FILE MANDATORY:genome fasta file default: none
-r FILE file containing list of filenames of reads from transcriptome sequencing experiments, default: none
FORMAT OF THIS FILE:
Each line in the file must refer to sequencing data from a single experiment.
Please combine runs so that one file/pair/triplet of files contains a single sample.
The lines are in the following format:
/path/filename /path/filename /path/filename tag
or
/path/filename /path/filename tag
or
/path/filename tag
Fields are space-separated, no leading space. "tag" indicates type of data referred to in the preceding fields. Possible values are:
fastq -- indicates the data is Illumina RNA-seq in fastq format, expects one or a pair of /path/filename.fastq before the tag
fasta -- indicates the data is Illumina RNA-seq in fasta format, expects one or a pair of /path/filename.fasta before the tag
bam -- indicates the data is aligned Illumina RNA-seq reads, expects one /path/filename.bam before the tag
bam_isoseq -- indicates the data is aligned PacBio Iso-seq reads, expects one /path/filename.bam before the tag
isoseq -- indicates the data is PacBio Iso-seq reads in fasta or fastq format, expects one /path/filename.(fasta or fastq) before the tag
mix -- indicates the data is from the sample sequenced with both Illumina RNA-seq provided in fastq format and long reads (Iso-seq or Oxford Nanopore) in fasta/fastq format, expects three /path/filename before the tag
bam_mix -- indicates the data is from the same sample sequenced with both Illumina RNA-seq provided in bam format and long reads (Iso-seq or Oxford Nanopore) in bam format, expects two /path/filename.bam before the tag
Absense of a tag assumes fastq tag and expects one or a pair of /path/filename.fastq on the line. If supplying the data as aligned BAM files, please make sure that the HISAT2 alignments were performed keeping information needed for StringTie (--dta option).
-e FILE fasta file with assembled transcripts from related species, default: none
-p FILE fasta file with protein sequences from (preferrably multiple) related species, uniprot proteins are used of this file is not provided, default: none
-m INT max intron size, default: 250000
-s FILE fasta file with UniProt-SwissProt proteins to use in functional annotation or if proteins from close relatives are not available. EviAnn uses a recent version of this protein database internally. To use the most up-to-date version, supply it with this switch. THe database is available at: https://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/uniprot_sprot.fasta.gz
-l liftover mode, optimizes internal parameters for annotation liftover; also useful when supplying proteins from a single species, default: not set
-f perform functional annotation, default: not set
--debug keep intermediate output files, default: not set
--verbose verbose run, default: not set
--version report version and exit, default: not set
-r or -e MUST be supplied.
EviAnn saves progress from all intermediate steps. If EviAnn run stops for any reason (computer rebooted or out of disk space), just re-run the same command and EviAnn will continue from the last successfuly completed stage.
EviAnn uses the input genome file name as <PREFIX> for intermediate/output files. If the input genome file is genome.fasta, then the <PREFIX> is "genome.fasta", and final annotation files are named genome.fasta.pseudo_label.gff, genome.fasta.proteins.fasta and genome.fasta.transcripts.fasta. These files contain annotation is GFF3 format, sequences of proteins (amino-acids) and transcripts.
EviAnn outputs the annotation in GFF3 format, along with translated protein sequences and transcripts in FASTA format. Per GFF3 convention, stop codon is included into the CDS. Every "mRNA" line for a protein coding transcript contains the following attributes:
- ID -- this is the transcript ID assigned by EviAnn
- Parent -- this is the ID of the parent feature
- EvidenceProteinID -- this is the ID of the protein that was used as evidence for the CDS annotation for this transcript. If the EvidenceProteinID starts with XLOC... then the transcript was annotated from the transcript alignment alone, please refer to the EvidenceTranscriptID for the evidence
- EvidenceTranscriptID -- this is the ID of the transcript that was used as evidence for the annotation for this transcript. All transcripts assembled from the evidence are listed in <PREFIX>.gtf. The EvidenceTrasncriptID can be a source protein ID if Evidence is "protein_only". For "complete" and "transcript_only" evidence, the format of the EvidenceTranscriptID is <transcript_name>:<number of RNA-seq experiments containing the transcript>:<maximum TPM>
- StartCodon -- this is the start codon in the CDS
- StopCodon -- this is the stop codon in the CDS
- Class -- this is the match class of the source protein alignment to the transcript; most reliable transcripts have class code of "=" or"k"
- Evidence -- this is the type of evidence that was used to annotate the transcript/CDS. Possible values are: "complete", meaning that both transcript and protein alignment data was used, "protein_only", meaning that the only protein alignment data was used and "transcript_only" meaning that only transcript data was used. For "transcript_only" evidence the CDS was derived with TransDecoder with subsequent confirmation by alignment to Uniprot database
- Optional: pseudo=true -- this tag is present if EviAnn designated the gene/transcript as processed pseudo gene. No CDS is output for these transcripts.
For long non-coding RNAs the "mRNA" line contains the following attributes:
- ID -- this is the transcript ID assigned by EviAnn
- Parent -- this is the ID of the parent feature
- EvidenceTranscriptID -- this is the ID of the transcript that was used as evidence for the annotation for this transcript. All transcripts assembled from the evidence are listed in <PREFIX>.gtf.
Here is an example of annotation of a protein coding gene, a pseudo-gene and a long non-coding RNA, with functional annotation:
NC_004353.4 EviAnn gene 29462 43759 . - . ID=XLOC_000048;geneID=XLOC_000048;type=protein_coding;Note=Similar to PLXNA2: Plexin-A2 (Homo sapiens);
NC_004353.4 EviAnn mRNA 32745 43754 . - . ID=XLOC_000048-mRNA-1;Parent=XLOC_000048;EvidenceProteinID=XP_001352289.2;EvidenceTranscriptID=MSTRG_00000148:4:7.702416;StartCodon=atg;StopCodon=TGA;Class==;Evidence=complete;Note=Similar to PLXNA2: Plexin-A2 (Homo sapiens);
NC_004353.4 EviAnn exon 32745 33125 . - . Parent=XLOC_000048-mRNA-1
NC_004353.4 EviAnn exon 33191 33373 . - . Parent=XLOC_000048-mRNA-1
NC_004353.4 EviAnn exon 35874 36457 . - . Parent=XLOC_000048-mRNA-1
NC_004353.4 EviAnn exon 36516 41285 . - . Parent=XLOC_000048-mRNA-1
NC_004353.4 EviAnn exon 42914 43754 . - . Parent=XLOC_000048-mRNA-1
NC_004353.4 EviAnn CDS 33018 33125 . - 0 Parent=XLOC_000048-mRNA-1
NC_004353.4 EviAnn CDS 33191 33373 . - 0 Parent=XLOC_000048-mRNA-1
NC_004353.4 EviAnn CDS 35874 36457 . - 2 Parent=XLOC_000048-mRNA-1
NC_004353.4 EviAnn CDS 36516 41285 . - 2 Parent=XLOC_000048-mRNA-1
NC_004353.4 EviAnn CDS 42914 43424 . - 0 Parent=XLOC_000048-mRNA-1
NC_004354.4 EviAnn gene 23461776 23464767 . - . ID=XLOC_001890;geneID=XLOC_001890;type=protein_coding;pseudo=true;Note=function unknown;
NC_004354.4 EviAnn mRNA 23461776 23464767 . - . ID=XLOC_001890-mRNA-1;Parent=XLOC_001890;EvidenceProteinID=XP_046868993.1;EvidenceTranscriptID=MSTRG_00006719:2:3.697180;StartCodon=atg;StopCodon=TAA;Class=k;Evidence=complete;pseudo=true;Note=function unknown;
NC_004354.4 EviAnn exon 23461776 23464767 . - . Parent=XLOC_001890-mRNA-1
NC_004353.4 EviAnn gene 181423 182801 . - . ID=XLOC_000055U_lncRNA;geneID=XLOC_000055U_lncRNA;type=lncRNA;junction_score=0;Note=function unknown;
NC_004353.4 EviAnn ncRNA 181423 182801 . - . ID=XLOC_000055U_lncRNA-mRNA-1;Parent=XLOC_000055U_lncRNA;EvidenceTranscriptID=MSTRG_00000163:5:11.129138
NC_004353.4 EviAnn exon 181423 181516 . - . Parent=XLOC_000055U_lncRNA-mRNA-1
NC_004353.4 EviAnn exon 181569 182801 . - . Parent=XLOC_000055U_lncRNA-mRNA-1
EviAnn produces annotations in a proper GFF3 format for ease of submission to NCBI Genbank. Here are the steps to take to produce NCBI compliant submission input files from EviAnn output:
- Fill out the form here to produce template.sbt file: https://submit.ncbi.nlm.nih.gov/genbank/template/submission/
- Download and install table2asn tool from NCBI: https://www.ncbi.nlm.nih.gov/genbank/table2asn/
- run this command in the EviAnn output folder:
table2asn -M n -J -c w -euk -t template.sbt -gaps-min 10 -l paired-ends -j "[organism=latin name][isolate=isolate]" -i assembly.fasta -f assembly.fasta.pseudo_label.gff -o output.sqn -Z -V b -locus-tag-prefix XXXX
This command assumes that the genome assembly fasta file is in the current folder and named "assembly.fasta", and thus the annotation file output by EviAnn is named "assembly.fasta.pseudo_label.gff". You can change the assembly name or path if it differs. XXXX is a four-letter locus tag prefix assigned by GenBank during assembly submission. This command produces output.sqn and output.gbf files that can be used to submit the annotation to GenBank.
Suppose that you are annotating genome sequence in genome.fasta. You have two pairs of RNA-seq files rna1_R1.fastq, rna1_R2.fastq, rna2_R1.fastq, rna2_R2.fastq, and protein sequences from several related species that you would like to use for annotation. The proteins from all related species must be in fasta format. Ideally you want about 5-10 times as many proteins from relates species as you expect to be present in the species you are annotating. For example, about 100000 - 200000 proteins for an insect, or about 500000 proteins for a typical plant genome or a typical mammalian genome. The individual files containing protein sequences must be concatenated into a single fasta file:
cat protein1.faa protein2.faa > proteins_all.faa
Next you need to create a file that lists all RNA-seq data (e.g. paired.txt here). This file must contain the names of the reads files with absolute or relative (v1.0.8 and up) paths, two per line, forward and then reverse, for example:
$ cat paired.txt
/path/rna1_R1.fastq /path/rna1_R2.fastq
/path/rna2_R1.fastq /path/rna2_R2.fastq
This file can be easily created by the following command (assuming you are in the folder where the RNA-seq data is located):
paste <(ls $PWD/*_R1.fastq) <(ls $PWD/*_R2.fastq) > paired.txt
Adjust wildcards in the above example to the names of your read files. If some of all of your RNA-seq data are in fasta format, or aligned in the bam format, you can use the fasta/BAM files and indicate that by adding "fasta" or "bam" tag as the last field on the line, e.g.:
$ cat paired_mixed.txt
/path/rna1_R1.fastq /path/rna1_R2.fastq /path/IsoSeq_rna.fastq
/path/rna1_R1.fastq /path/rna1_R2.fastq
/path/rna2_R1.fa /path/rna2_R2.fa fasta
/path/rna3.bam bam
it is important to specify all input files to EviAnn with absolute paths if you are using a version earlier than 1.0.8. If you wish to run EviAnn with 24 threads, you can now run EviAnn as follows:
/path/EviAnn-X.X.X/bin/eviann.sh -t 24 -g /path/genome.fasta -r /path/paired.txt -p /path/proteins_all.faa
Substitute EviAnn version number for the X's.
Suppose again that you are annotating genome sequence in genome.fasta. In this scenario we assume that you have gff files containing the annotations of the related species that you are going to use as evidence. This scenario can also be descibed as "lifting over" annotation from one or more related species. The genome sequences for these species are also needed. The first step is to create transcripts and proteins files for each species with the following command:
/eviann_path/bin/gffread -W -y species1_prot.faa -w species1_transc.fa -g species1_genome.fa species1.gff
/eviann_path/bin/gffread -W -y species2_prot.faa -w species2_transc.fa -g species2_genome.fa species2.gff
etc...
The next step is to concatenate all proteins files and all transcript files into a single file:
cat species*_transc.fa > transcripts.fa
cat species*_prot.fa > proteins.faa
Then you can run EviAnn with 24 threads (for example) as follows:
/path/EviAnn-X.X.X/bin/eviann.sh -t 24 -g /path/genome.fasta -e $PWD/transcripts.fa -p $PWD/proteins.faa -l
Make sure that you use -l switch! This will optimize internal parameters for liftover run. Substitute EviAnn version number for the X's.
1. Here are the steps you can follow to create and download protein evidence file from NCBI. Go to https://www.ncbi.nlm.nih.gov/taxonomy:
3. NCBI will find the lineage and species name. First try using the rightmost link in the lineage list (Malus). If the subsequent steps result in fewer than 150,000 protein hits, you can move up to the next available lineage level on the left (in this case Maleae).
5. Look for the red "Protein" word in the table on the upper right. If the number to the right of the link is > 150,000, click on the number, otherwise go back to step 3 and choose lineage that is higher up in the tree. For best results I recommend usng proteins from at least five related species.
6. Click "Send to", choose "File" format "FASTA", and click "Create File" button. Save the file as "proteins.faa". You can use this file as input proteins to EviAnn ( -r proteins.faa ).
