Michael G. Campana and Madeline W. Eibner-Gebhardt, 2023-2024
Smithsonian's National Zoo and Conservation Biology Institute
Pipeline to identify Avipoxvirus sequences in DNA libraries
Eibner-Gehbardt MW, Fleischer RC, Campana MG. In prep. A historical Hawaiian Avipoxvirus genome reconstructed from an 1898 museum specimen.
The software is made available under the Smithsonian Institution terms of use.
The pipeline requires a directory of bidirectionally sequenced DNA libraries in FASTQ format. The pipeline expects the forward and reverse reads in separate files. Second, the pipeline requires a CSV file assigning the read files to libraries with the following format:
Library,Read1,Read2,Adapter1,Adapter2
<library1 name>,<path to library1 read1>,<path to library1 read2>,<library1 adapter1 sequence>,<library1 adapter2 sequence>
<library2 name>,<path to library2 read1>,<path to library2 read2>,<library2 adapter1 sequence>,<library2 adapter2 sequence>
Additionally, the pipeline requires access to a local copy of the NCBI non-redundant nucleotide database ('nt') as well as a custom Avipoxvirus genomic blast database. See Eibner-Gehbardt et al. for details on compiling the Avipoxvirus genome database.
The pipeline requires Nextflow. The pipeline can automatically install the dependencies using Conda. After installing Nextflow and Conda, install the pipeline with the command:
nextflow pull campanam/Avipoxvirus -r main
If you cannot install the dependencies automatically using the Conda, please install the following software packages:
AdapterRemoval v. 2.3.3 (1)
Seqtk v. 1.4 (2)
CD-HIT v. 4.8.1 (3)
BLAST+ v. 2.13.0 (4)
MEGAN - Community Edition v. 6.24.20 (5)
We recommend copying and modifying the nextflow.config file included in this directory. A profile ('hydra') describing the configuration and Conda recipes used in Eibner-Gebhardt et al. is included in the nextflow.config file. Please consult the Nextflow documentation to configure the pipeline for your system.
Configurable parameters needed for the pipeline are:
inputCsv: Path to the input CSV of Samples
blastdb: Path to custom Avipoxvirus BLAST database
ntdb: Path to BLAST nt database
readsdir: Path to directory holding raw read pairs
outdir: Output directory
outstem: Output file stem
taxon: Taxon to search for in MEGAN LCA files
Enter the command:
nextflow run campanam/Avipoxvirus -r main -c <your config file>
The pipeline will create the and populate the following output subdirectories:
01_BlastResults: Unique, merged sequences in FASTA format and their BLAST results against the custom Avipoxvirus database in XML format.
02_RMA_LCA: MEGAN RMA6 and LCA files derived from BLAST XML results. Files derived from the second BLAST step against the nt database will have '_avi' appended before the file suffix (either .lca.txt or .rma6).
03_BlastHits: Reads that match Avipoxvirus in the first round of BLAST analysis in FASTA format.
04_Summary: A summary file of the number of Avipoxvirus hits per library after the first round of BLAST assignment.
05_NtBlastResults: BLAST results in XML format after secondary assignment against nt database.
06_NtSummary: A summary file of the number of Avipoxvirus hits per library after the second round of BLAST assignment.
- Schubert M, Lindgreen S, Orlando L. 2016. AdapterRemoval v2: rapid adapter trimming, identification, and read merging. BMC Research Notes. 9: 88.
- Li H. 2023. Seqtk v. 1.4. Available: https://github.com/lh3/seqtk.
- Li W, Godzik A. 2006. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 22: 1658-9.
- Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLAST+: architecture and applications. BMC Bioinformatics. 10: 421.
- Huson DH, Beier S, Flade I, Górska M, Mitra S, Ruscheweyh H-J, Tappu R. 2016. MEGAN Community Edition - interactive exploration and analysis of large-scale microbiome sequencing data. PLoS Computational Biology. 12: e1004957.