The Pipeline needs to be configured to match your local compute environment and to set parameters specific to your assembly and read data. These configuration options are set in a YAML format file before the Pipeline is run. The Pipeline has been designed for use with publicly available assemblies, but most configuration options are equally applicable for use with local datasets. Starting with a configuration file for a publicly available Drosophila albomicans assembly, the configuration is divided into six sections:
assembly: accession: GCA_000298335.1 alias: DroAlb_1.0 bioproject: PRJNA39511 biosample: SAMN00003213 level: scaffold scaffold-count: 26354 span: 253560284 prefix: ACVV01 busco: lineages: - diptera_odb9, - arthropoda_odb9 - eukaryota_odb9 lineage_dir: /path/to/busco/lineages reads: paired: - - SRR026696 - ILLUMINA - 482114248 - ftp.sra.ebi.ac.uk/vol1/fastq/SRR026/SRR026696/SRR026696_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR026/SRR026696/SRR026696_2.fastq.gz - - SRR026697 - ILLUMINA - 552054360 - ftp.sra.ebi.ac.uk/vol1/fastq/SRR026/SRR026697/SRR026697_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR026/SRR026697/SRR026697_2.fastq.gz single:  coverage: max: 100 min: 0.5 settings: blobtools2_path: /path/to/blobtoolkit/blobtools2 taxonomy: /path/to/blobtoolkit/taxdump/ tmp: /tmp blast_chunk: 100000 blast_max_chunks: 10 blast_overlap: 500 chunk: 1000000 similarity: defaults: evalue: 1e-25 max_target_seqs: 10 root: 1 mask_ids: - 7215 databases: - local: /path/to/databases/ncbi_2019_08 name: nt source: ncbi tool: blast type: nucl - local: /ceph/software/databases/uniprot_2019_07 max_target_seqs: 1 name: reference_proteomes source: uniprot tool: diamond type: prot taxrule: bestsumorder taxon: taxid: 7291 name: Drosophila albomicans keep_intermediates: true
assembly: accession: GCA_000298335.1 alias: DroAlb_1.0 bioproject: PRJNA39511 biosample: SAMN00003213 level: scaffold scaffold-count: 26354 span: 253560284 prefix: ACVV01
assembly section for a publicly available assembly contains details of the assembly GCA
alias to allow the assembly FASTA to be retrieved from NCBI. Assembly
biosample are included to allow direct links to the source data from within the BlobToolKit Viewer. Assembly
level can be set to scaffold, contig or chromosome depending on the top level to which the genome has been assembled. The
span provide metadata that can be checked against the values obtained when importing the assembly and
span can be used to determine read coverage prior to mapping. Finally the
prefix is set to the assembly WGS accession by convention and is used to determine the assembly filename. For local assemblies many of these fields may be omitted and the prefix should be set to the basename of your assembly FASTA file, e.g. if this were a local assembly saved as
assembly: accession: draft level: scaffold scaffold-count: 26354 span: 253560284 prefix: Dalbomicans_v1
busco: lineages: - diptera_odb9, - arthropoda_odb9 - eukaryota_odb9 lineage_dir: /path/to/busco/lineages
Running BUSCO analyses as part of the Pipeline is optional, but the section should be present in either case. A separate BUSCO analysis will be run for each of the listed
lineages. If any of the listed lineages are not already available in the
lineage_dir, they will be fetched automatically when the Pipeline is run. Valid lineage names are any lineages available from the BUSCO website. To run the Pipeline without any BUSCO analyses, the list of lineages should be left empty and a minimal
busco section would be:
busco: lineage_dir: /path/to/busco/lineages lineages: 
reads: paired: - - SRR026696 - ILLUMINA - 482114248 - ftp.sra.ebi.ac.uk/vol1/fastq/SRR026/SRR026696/SRR026696_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR026/SRR026696/SRR026696_2.fastq.gz - - SRR026697 - ILLUMINA - 552054360 - ftp.sra.ebi.ac.uk/vol1/fastq/SRR026/SRR026697/SRR026697_1.fastq.gz;ftp.sra.ebi.ac.uk/vol1/fastq/SRR026/SRR026697/SRR026697_2.fastq.gz single:  coverage: max: 100 min: 0.5
reads section contains information on
single ended read files to be mapped to the genome. Either or both of these sections may be present, depending on the read files available for mapping, and both follow the same pattern. Each read file is represented by an array of up to four values: (i) name, (ii) sequencing platform, (iii) base count and (iv) remote file URL(s). For local files, only the first two values are required.
(i) For publicly available assemblies, the name should be the SRA accession and must match the accession in the remote file URL. For local assemblies the name should match the basename of your read FASTQ file. Local filenames must match the pattern
name.fastq.gz for single end reads,
name_subreads.fastq.gz for PacBio reads or
name_2.fastq.gz for paired end files.
(ii) The sequencing platform is used to set the appropriate parameters for read mapping using Minimap2. Valid values are “ILLUMINA”, “OXFORD_NANOPORE”, “PACBIO_SMRT” and “LS454”.
(iii) If set, the base count value is used in conjunction with the
coverage subsection described below to determine whether read files should be subsampled prior to mapping.
(iv) For public assemblies, the remote file URL(s) from which the read files can be obtained must be set so they can be fetched by the Pipeline. for
paired files, the two URLs should be separated by a semicolon as in the example above.
coverage subsection allows
min coverage values to be set based on base count values provided in the
single subsections. Files with a coverage (base count /
assembly.span) greater than
max will be subsampled using seqtk, while files with less than
min coverage will not be mapped.
To map all reads in local PacBio read files named
library2_subreads.fastq.gz, regardless of coverage, the reads section would look like:
reads: single: - - library1 - PACBIO_SMRT - - library2 - PACBIO_SMRT
To map all reads in local paired Illumina read files named readfile_1.fastq.gz and readfile_2.fastq.gz, the reads section would look like:
reads: paired: - - readfile - ILLUMINA
settings: blobtools2_path: /path/to/blobtoolkit/blobtools2 taxonomy: /path/to/databases/taxonomy_2019_08 tmp: /tmp blast_chunk: 100000 blast_max_chunks: 10 blast_overlap: 500 chunk: 1000000
Configuration options in the
settings section are used to set some program/file locations that are not handled elsewhere and a number of parameters for the BLAST wrapper script. Most Pipeline dependencies are handled using Conda environments, however the
blobtools2_path must be set to the location of this program on your local system.
The Pipeline requires a local copy of the NCBI taxonomy new_taxdump, which will be automatically fetched and added to the
taxonomy directory. This file should match the taxonomy used in the
nt database (see below) so it is useful to keep an explicit record of the date downloaded in the directory name. Finally, some database preparation steps can use a large amount of disk space so a suitable
tmp directory should be specified.
The remaining parameters set defaults that need to be included in the file but will not normally need to be changed. These mostly relate to the BLAST wrapper script that splits long scaffold sequences into chunks before running BLAST searches to avoid subsequent taxonomic inference being based on a single region within the sequence.
blast_chunk value determines a minimum length of the chunks that long scaffolds will be split into by the BLAST wrapper script.
blast_max_chunks ensures that very long scaffolds will only be split into a limited number of chunks, for scaffolds longer than
blast-max_chunks, the chunk length will be increased so all subsequences are of equal size. The
blast_overlap value sets a small overlap between chunks to allow for hits that span the break points. (The similarly named
chunk parameter is unrelated and should no longer be required but this needs testing.)
similarity: defaults: evalue: 1e-25 max_target_seqs: 10 root: 1 mask_ids: - 7215 databases: - local: /path/to/databases/ncbi_2019_08 name: nt source: ncbi tool: blast type: nucl - local: /ceph/software/databases/uniprot_2019_07 max_target_seqs: 1 name: reference_proteomes source: uniprot tool: diamond type: prot taxrule: bestsumorder
similarity section controls the databases and settings used for sequence similarity searches. The structure of this section reflects a slightly more flexible approach to database specification than is currently in use.
defaults are settings applied to all sequence similarity searches and in the example file includes the
max_target_seqs values. The
root value is the NCBI Taxonomy ID of the root of the clade that you wish to search against. For analyses of public datasets we always set this to 1 to include all taxa but it provides the option to limit a search to a single kingdom or phylum when analysing local assemblies.
mask_ids is a list of Taxonomy IDs to for which all descendants will be excluded from the similarity searches. For the analysis of public assemblies we set this to the genus of the taxon being analysed (7215 is Drosophila) to avoid assigning taxonomy based on the data in the assembly that may already be in the database we are searching against. For analyses of local assemblies this should usually be set to be an empty list (i.e.
databases subsection is a list of database-specific parameters.
local is the path to a local copy of the database (that will be fetched/generated if it does not already exist).
name is the database name, for NCBI BLAST searches this must match
nt and be a version 5 database to support the taxonomy-based filtering discussed above.
source is either “ncbi” or “uniprot” and determines where the database files should be fetched from.
tool is either “blast” for NCBI BLAST or “diamond” and
type is “nucl” for nucleotide or “proc” for protein. The flexibility that this implies has not been tested recently so it is best to leave all but
taxrule specifies which BlobTools2 taxrule should be used to assign taxonomic labels to the assembly scaffolds. In practice, this should always be set to “bestsumorder” as Diamond searches are only performed for scaffolds with no NCBI BLAST hits.
taxon: taxid: 7291 name: Drosophila albomicans
taxon section should contain a NCBI
taxid to allow full taxonomic information to be added to the dataset metadata. The taxon
name should be the species, subspecies or strain name, as appropriate.
keep_intermediates is a flag that should be set to “true” if you wish to keep all intermediate files generated by the pipeline. When running the pipeline on public assemblies we set this flag to “false” so some large files are discarded once they have been used. For local assemblies, these files (e.g. read-alignment BAM files) are likely to be useful for further analyses.