Protein ALignment And Detection INterface
PALADIN is a protein sequence alignment tool designed for the accurate functional characterization of metagenomes.
PALADIN is based on BWA, and aligns sequences via read-mapping using BWT. PALADIN, however, offers the novel approach of aligning in the protein space. During the index phase, it processes the reference genome's nucleotide sequences and GTF/GFF annotation containing CDS entries, first converting these transcripts into the corresponding protein sequences, then creating the BWT and suffix array from these proteins. The process of translatation is skiped when providing a protein reference file (e.g., UniProt) for mapping. During the alignment phase, it attempts to find ORFs in the read sequences, then converts these to protein sequences, and aligns to the reference protein sequences.
PALADIN currently only supports single-end reads (or reads merged with FLASH, PEAR, abyss-mergepairs), and BWA-MEM based alignment. It makes use of many BWA parameters and is therefore compatible with many of its command line arguments.
PALADIN may output a standard SAM file, or a text file containing a UniProt-generated functional profile. This text file may be used for all downstream characterizations.
- From a fresh install of Ubuntu, you will need to install
build-essential libcurl4-openssl-dev git make gcc zlib1g-dev. This should be available on Ubuntu 14.04 using
sudo apt-get install build-essential libcurl4-openssl-dev git make gcc zlib1g-dev
- PALADIN compiles by default on OSX 10.10.x
git clone https://github.com/twestbrookunh/paladin.git cd paladin/ make
Alternatively, you can use Paladin with the Docker image hosted at https://quay.io/repository/fhcrc-microbiome/paladin. This image can be downloaded with the command
docker pull quay.io/fhcrc-microbiome/paladin. A set of tags are used to pin releases, e.g.
v1.4.0--1 is the image pinned to the
v1.4.0 version of Paladin.
Download and prepare UniProt Swiss-Prot index files.
paladin prepare -r1
Download and prepare UniProt UniRef90 index files.
paladin prepare -r2
Index UniProt (or another protein) fasta. (See here for an explanation of
paladin index -r3 uniprot_sprot.fasta.gz
Align a set of reads using 4 theads. Send the full UniProt report to paladin_uniprot.tsv.
paladin align -t 4 -o paladin index input.fastq.gz
Align a set of reads using 4 theads. Produce a bam file.
paladin align -t 4 index input.fastq.gz | samtools view -Sb - > test.bam
Align a set of reads, preferring higher quality mappings over number of proteins detected.
paladin align -T 20 -o paladin index input.fastq.gz
Align a set of reads, report secondary alignments, and generate UniProt report for both primary and secondary alignments.
paladin align -a -o paladin index input.fastq.gz
If you're intersted in trying this out on a smallish test file, try downloading this one which is from a human lung metagenome study: http://www.ebi.ac.uk/ena/data/view/PRJNA71831
#install PALADIN as per above curl -O ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR117/002/SRR1177122/SRR1177122.fastq.gz paladin prepare -r1 #unless already done paladin align -t 4 -o lungstudy uniprot_sprot.fasta.gz SRR1177122.fastq.gz #look at report file, SAM, etc.
All references must be initially indexed via either the
index commands before use with PALADIN.
prepare command must be run for all UniProt or UniProt formatted references, and is necessary if you wish PALADIN to generate the TSV UniProt characterization report during alignment. The
prepare command will download and index the Swiss-Prot or UniRef90 protein databases by default, or you may specify a local, custom reference formatted using the
>sp|AccID|KBID header format of the Swiss-Prot database, using valid UniProt KBIDs.
If you do not require the TSV file during alignment, and only wish to generate SAM alignment data, the
index command is sufficient to index the reference. References indexed via the
index command do not need to adhere to the UniProt format. References indexed via the
index command may later be upgraded to fully prepared references using
prepare, as long as they adhere to the UniProt header format.
This repo also contains a wrapper script (
run.py) which is intended to make it easier to deploy Paladin on cloud or HPC computing resources (e.g. Slurm or AWS). The script is located in the PATH in the Docker image, and so you can run
run.py -h to see the set of options for this script. In brief, users can specify the input URL, reference database path, and output folder location (any of which may be local paths, S3 buckets, or FTP). The run script will fetch the input data, run Paladin, wrap up the results into a single JSON output file, and copy the results to the specified output folder.
- A SAM/BAM file that can be used for any downstream analyses.
- A tab delimited UniProt report file.
Note: Be sure to filter your results by the maximum mapping quality. Though an ORF may successfully map to a protein/cluster, this does not indicate how likely this is a correct mapping. Without filtering, the SAM/TSV files will include may entries that are likely noise. A high maximum mapping quality indicates at least one ORF mapped with high confidence to the reported protein/cluster.
TSV FORMAT Count Abundance Quality (Avg) Quality (Max) UniProtKB ID Organism Protein Names Genes Pathway Features Gene Ontology Reviewd Existence Comments Cross Reference (KEGG) Cross Reference (GeneID) Cross Reference (PATRIC) Cross Reference(EnsemblBacteria)
- Count: The number of reads mapping to that UniProt entry
- Abundance: The percentage of reads mapping to that UniProt entry
- Quality (Avg): The average mapping quality for reads mapped to that UniProt entry (Phred scale, max 60)
- Quality (Max): The maximum mapping quality for reads mapped to that UniProt entry (Phred scale, max 60)
- UniProtKB: The ID containing the Gene short-code and species of origin
- ID: The Uniprot code
- Organims: The Organims from which the Uniprot ID is derived. Note that one should use this to generate a taxonomic profile of your sample
- Protein Names
- Pathway Features
- Gene Ontology
- Cross Reference (KEGG): Corresponding entry in KEGG database (http://www.genome.jp/kegg/)
- Cross Reference (GeneID): Corresponding entry in NCBI gene database (http://www.ncbi.nlm.nih.gov/gene)
- Cross Reference (PATRIC): Corresponding entry in PATRIC database (http://www.patricbrc.org)
- Cross Reference (EnsemblBacteria): Corresponding entry in Ensembl Bacteria database (http://bacteria.ensembl.org)
A number of preparation and downstream analysis tools are available via the PALADIN-plugins package. This includes HPC MPI support, customized UniProt reports, taxonomic analysis, GO term analysis, etc. PALADIN-plugins can be found here.