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isugifNF/assemblyStats v1.0.0
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isugifNF/assemblyStats is a nextflow pipeline to assess the quality of your genome assembl(y/ies). It runs three separate programs
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I found AssemblathonStats formatting to not be to my liking so I rewrote the script to output the same statistics but in an easier to read format with a few additional outputs such as the top 5 largest and smallest scaffolds
Example
``` Number of Scaffolds: 3 Total Nucleotide content 4658411 Longest Scaffold: 4553770 >NC_017168.1 Yersinia pestis A1122, complete sequence Shortest Scaffolds: 8431 >NC_017170.1 Yersinia pestis A1122 plasmid unnamed, complete sequence Mean Scaffold Size 1552803 Median Scaffold length 96210.0 N50 Scaffold length 4553770 L50 Scaffold length 1 N90 Scaffold length 4553770 L90 Scaffold length 1 #Scaffs % Scaffolds Nucleotides % Nucleotide Content Number of Scaffolds [0-1K) nt 0 0.0 % 0 0.0 % Number of Scaffolds [1K-10K) nt 1 33.33 % 8431 0.180 % Number of Scaffolds [10K-100K) nt 1 33.33 % 96210 2.065 % Number of Scaffolds [100K-1M) nt 0 0.0 % 0 0.0 % Number of Scaffolds [1M-10M) nt 1 33.33 % 4553770 97.75 % Number of Scaffolds > 10M nt 0 0.0 % 0 0.0 % ```
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Technically this is a modified version of this script that includes N90/L90 as well.
Example
``` ---------------- Information for assembly 'Ecoli.fasta' ---------------- Number of scaffolds 3 Total size of scaffolds 4658411 Longest scaffold 4553770 Shortest scaffold 8431 Number of scaffolds > 1K nt 3 100.0% Number of scaffolds > 10K nt 2 66.7% Number of scaffolds > 100K nt 1 33.3% Number of scaffolds > 1M nt 1 33.3% Number of scaffolds > 10M nt 0 0.0% Mean scaffold size 1552804 Median scaffold size 96210 N50 scaffold length 4553770 L50 scaffold count 1 n90 scaffold length 4553770 L90 scaffold count 1 scaffold %A 26.07 scaffold %C 23.69 scaffold %G 23.94 scaffold %T 26.30 scaffold %N 0.00 scaffold %non-ACGTN 0.00 Number of scaffold non-ACGTN nt 0 Percentage of assembly in scaffolded contigs 0.0% Percentage of assembly in unscaffolded contigs 100.0% Average number of contigs per scaffold 1.0 Average length of break (>25 Ns) between contigs in scaffold 0 Number of contigs 3 Number of contigs in scaffolds 0 Number of contigs not in scaffolds 3 Total size of contigs 4658411 Longest contig 4553770 Shortest contig 8431 Number of contigs > 1K nt 3 100.0% Number of contigs > 10K nt 2 66.7% Number of contigs > 100K nt 1 33.3% Number of contigs > 1M nt 1 33.3% Number of contigs > 10M nt 0 0.0% Mean contig size 1552804 Median contig size 96210 N50 contig length 4553770 L50 contig count 1 n90 contig length 4553770 L90 contig count 1 contig %A 26.07 contig %C 23.69 contig %G 23.94 contig %T 26.30 contig %N 0.00 contig %non-ACGTN 0.00 Number of contig non-ACGTN nt 0 ```
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BUSCO Orthologs
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Benchmarking Universal Single Copy
Example
***** Results:***** C:100.0%[S:100.0%,D:0.0%],F:0.0%,M:0.0%,n:124 124 Complete BUSCOs (C) 124 Complete and single-copy BUSCOs (S) 0 Complete and duplicated BUSCOs (D) 0 Fragmented BUSCOs (F) 0 Missing BUSCOs (M) 124 Total BUSCO groups searched
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Run Assembly statistics on a genome assembly (BUSCO and assemblyStats and new_Assemblathon.pl)
Assuming the system already has nextflow and singularity, running the following should automatically pull the pipeline.
nextflow run isugifNF/assemblyStats --help
see usage statement
Usage:
The typical command for running the pipeline are as follows:
nextflow run isugifNF/assemblyStats --genomes "*fasta" --outdir newStats3 --threads 16 --options "-l eukaryota_odb10" -profile condo,singularity
nextflow run isugifNF/assemblyStats --genomes "*fasta" --outdir newStats3 --threads 16 --options "-l mollusca_odb10" -profile ceres,singularity --buscoOnly
Mandatory arguments:
--genomes genome assembly fasta files to run stats on. (./data/*.fasta)
-profile singularity (docker) as of now, this workflow only works using singularity or docker and requires this profile [be sure singularity is in your path or loaded by a module]
Optional arguments:
--outdir Output directory to place final output
--threads Number of CPUs to use during the NanoPlot job [default:40]
--queueSize Maximum number of jobs to be queued [default:18]
--options [default:'--auto-lineage'], you may also consider "--auto-lineage-prok","--auto-lineage-euk", "-l eukaryota_odb10"
--listDatasets Display the list of available BUSCO lineage datasets to use in --options pipeline parameter.
--buscoOnly When you just want to run a different lineage and not rerun the assemblathon stats
--account Some HPCs require you supply an account name for tracking usage. You can supply that here.
--help This usage statement.
Programs required for this workflow are included in two singularity containers docker://swift and docker://ezlabgva/busco. These containers should be automatically pulled by nextflow. (Will only need to run singularity pull if website connection is unstable. On HPC machines, the singularity pull may time out, in which case we recommend manually pulling the singularity images and placing it in the work/singularity folder.
Manually pulling the singuarlity images
Run the following to manually pull the two singularity containers. If on the HPC, you may need to be on the dtn node.
singularity pull --name swift.img docker://swift > /dev/null
singularity pull --name ezlabgva-busco-v5.1.2_cv1.img docker://ezlabgva/busco:v5.1.2_cv1 > /dev/null
ls *.img
#> ezlabgva-busco-v5.1.2_cv1.img swift.img
When you attempted to run this pipeline, Nextflow should have created a work/singularity folder in the current directory. If not, create it and add the singularity images.
mkdir -p work/singularity
mv *.img work/singularity/.
Download a dataset of Bacteria
#E. coli
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/Yersinia_pestis/representative/GCF_000222975.1_ASM22297v1/GCF_000222975.1_ASM22297v1_genomic.fna.gz
mv GCF_000222975.1_ASM22297v1_genomic.fna.gz Ecoli.fasta.gz
gunzip Ecoli.fasta.gz
#Yersinia Pestis
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/Escherichia_coli/reference/GCF_000005845.2_ASM584v2/GCF_000005845.2_ASM584v2_genomic.fna.gz
mv GCF_000005845.2_ASM584v2_genomic.fna.gz Yerpes.fasta.gz
gunzip Yerpes.fasta.gz
Run Nextflow. I am using singularity on my remote HPC called Nova. This repo contains config files for different machines for how to submit using the SLURM scheduler.
nextflow run isugifNF/assemblyStats \
--genomes "*.fasta" \
--options "-l bacteria_odb10" \
-profile singularity,nova
The output directory default is out_dir and can be changed by specifying the --outdir parameter on the command line. It contains
| Folder | Description |
|---|---|
| BUSCO | soft links to BUSCO output files, logs and blast results |
| BUSCOResults | short_summary.specific final output |
| assemblathonStats | text file of assemblathon statistics for the genome |
| assemblyStats | text file of assemblyStats statistics for the genome |
| Busco_version.txt | Version of BUSCO used in the container |
Nextflow is written in groovy which requires java version 1.8 or greater (check version using java -version). But otherwise can be installed if you have a working linux command-line.
java -version
curl -s https://get.nextflow.io | bash
# Check to see if nextflow is created
ls -ltr nextflow
#> total 32
#> -rwx--x--x 1 username staff 15K Aug 12 12:47 nextflow
First let's look at the help message
nextflow run isugifNF/assemblyStats --help
Output
N E X T F L O W ~ version 20.07.1
Launching `isugifNF/assemblyStats/main.nf` [magical_colden] - revision: a156628d62
----------------------------------------------------
\\---------//
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| (___ | | / _ | |_ \-//
_|_ ___) |__| \_/ _|_ | // \
//-----\\
//---------\\
isugifNF/nanoQCtrim v1.0.0
----------------------------------------------------
Usage:
The typical command for running the pipeline are as follows:
nextflow run isugifNF/assemblyStats --genomes "*fasta" --outdir newStats3 --threads 16 --options "-l eukaryota_odb10" -profile condo,singularity
nextflow run isugifNF/assemblyStats --genomes "*fasta" --outdir newStats3 --threads 16 --options "-l mollusca_odb10" -profile condo,singularity --buscoOnly
Mandatory arguments:
--genomes genome assembly fasta files to run stats on. (./data/*.fasta)
-profile singularity (docker) as of now, this workflow only works using singularity or docker and requires this profile [be sure singularity is in your path or loaded by a module]
Optional arguments:
--outdir Output directory to place final output
--threads Number of CPUs to use during the NanoPlot job [16]
--queueSize Maximum number of jobs to be queued [18]
--options ["--auto-lineage"], you may also consider "--auto-lineage-prok","--auto-lineage-euk",""-l eukaryota_odb10"
--listDatasets Display the list of available BUSCO lineage datasets to use in --options pipeline parameter.
buscoOnly When you just want to run a different lineage and not rerun the assemblathon stats
--help This usage statement.
We can get a list of the BUSCO datasets we can run using this set of parameters. The -profile docker is important as this workflow relies on containers and will error out if you don't use docker or singularity
nextflow run isugifNF/assemblyStats --listDatasets -profile docker
Output
N E X T F L O W ~ version 20.07.1
Launching `isugifNF/assemblyStats/main.nf` [amazing_colden] - revision: a156628d62
executor > local (1)
[6c/31848c] process > runBUSCOlist [ 0%] 0 of 1
INFO: Downloading information on latest versions of BUSCO data...
INFO: Downloading file 'https://busco-data.ezlab.org/v4/data/information/lineages_list.2019-11-27.txt.tar.gz'
INFO: Decompressing file '/Users/severin/work/6c/31848cd8f040c93f4047d085609d69/busco_downloads/information/lineages_list.2019-11-27.txt.tar.gz'
################################################
Datasets available to be used with BUSCOv4 as of 2019/11/27:
bacteria_odb10
- acidobacteria_odb10
- actinobacteria_phylum_odb10
- actinobacteria_class_odb10
- corynebacteriales_odb10
- micrococcales_odb10
- propionibacteriales_odb10
- streptomycetales_odb10
- streptosporangiales_odb10
- coriobacteriia_odb10
- coriobacteriales_odb10
- aquificae_odb10
- bacteroidetes-chlorobi_group_odb10
- bacteroidetes_odb10
- bacteroidia_odb10
- bacteroidales_odb10
- cytophagia_odb10
- cytophagales_odb10
- flavobacteriia_odb10
- flavobacteriales_odb10
- sphingobacteriia_odb10
- chlorobi_odb10
- chlamydiae_odb10
- chloroflexi_odb10
- cyanobacteria_odb10
- chroococcales_odb10
- nostocales_odb10
- oscillatoriales_odb10
- synechococcales_odb10
- firmicutes_odb10
- bacilli_odb10
- bacillales_odb10
- lactobacillales_odb10
- clostridia_odb10
- clostridiales_odb10
- thermoanaerobacterales_odb10
- selenomonadales_odb10
- tissierellia_odb10
- tissierellales_odb10
- fusobacteria_odb10
- fusobacteriales_odb10
- planctomycetes_odb10
- proteobacteria_odb10
- alphaproteobacteria_odb10
- rhizobiales_odb10
- rhizobium-agrobacterium_group_odb10
- rhodobacterales_odb10
- rhodospirillales_odb10
- rickettsiales_odb10
- sphingomonadales_odb10
- betaproteobacteria_odb10
- burkholderiales_odb10
- neisseriales_odb10
- nitrosomonadales_odb10
- delta-epsilon-subdivisions_odb10
- deltaproteobacteria_odb10
- desulfobacterales_odb10
- desulfovibrionales_odb10
- desulfuromonadales_odb10
- epsilonproteobacteria_odb10
- campylobacterales_odb10
- gammaproteobacteria_odb10
- alteromonadales_odb10
- cellvibrionales_odb10
- chromatiales_odb10
- enterobacterales_odb10
- legionellales_odb10
- oceanospirillales_odb10
- pasteurellales_odb10
- pseudomonadales_odb10
- thiotrichales_odb10
- vibrionales_odb10
- xanthomonadales_odb10
- spirochaetes_odb10
- spirochaetia_odb10
- spirochaetales_odb10
- synergistetes_odb10
- tenericutes_odb10
- mollicutes_odb10
- entomoplasmatales_odb10
- mycoplasmatales_odb10
- thermotogae_odb10
- verrucomicrobia_odb10
archaea_odb10
- thaumarchaeota_odb10
- thermoprotei_odb10
- thermoproteales_odb10
- sulfolobales_odb10
- desulfurococcales_odb10
- euryarchaeota_odb10
- thermoplasmata_odb10
- methanococcales_odb10
- methanobacteria_odb10
- methanomicrobia_odb10
- methanomicrobiales_odb10
- halobacteria_odb10
- halobacteriales_odb10
- natrialbales_odb10
- haloferacales_odb10
eukaryota_odb10
- alveolata_odb10
- apicomplexa_odb10
- aconoidasida_odb10
- plasmodium_odb10
- coccidia_odb10
- euglenozoa_odb10
- fungi_odb10
- ascomycota_odb10
- dothideomycetes_odb10
- capnodiales_odb10
- pleosporales_odb10
- eurotiomycetes_odb10
- chaetothyriales_odb10
- eurotiales_odb10
- onygenales_odb10
- leotiomycetes_odb10
- helotiales_odb10
- saccharomycetes_odb10
- sordariomycetes_odb10
- glomerellales_odb10
- hypocreales_odb10
- basidiomycota_odb10
- agaricomycetes_odb10
- agaricales_odb10
- boletales_odb10
- polyporales_odb10
- tremellomycetes_odb10
- microsporidia_odb10
- mucoromycota_odb10
- mucorales_odb10
- metazoa_odb10
- arthropoda_odb10
- arachnida_odb10
- insecta_odb10
- endopterygota_odb10
- diptera_odb10
- hymenoptera_odb10
- lepidoptera_odb10
- hemiptera_odb10
- mollusca_odb10
- nematoda_odb10
- vertebrata_odb10
- actinopterygii_odb10
- cyprinodontiformes_odb10
- tetrapoda_odb10
- mammalia_odb10
- eutheria_odb10
- euarchontoglires_odb10
- glires_odb10
- primates_odb10
- laurasiatheria_odb10
- carnivora_odb10
- cetartiodactyla_odb10
- sauropsida_odb10
- aves_odb10
- passeriformes_odb10
- stramenopiles_odb10
- viridiplantae_odb10
- chlorophyta_odb10
- embryophyta_odb10
- liliopsida_odb10
- poales_odb10
- eudicots_odb10
- brassicales_odb10
- fabales_odb10
- solanales_odb10
executor > local (1)
[6c/31848c] process > runBUSCOlist [100%] 1 of 1 ✔
Let's use a couple of small Bacterial genome for the test. So we will use bacteria_odb10
#E. coli
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/Yersinia_pestis/representative/GCF_000222975.1_ASM22297v1/GCF_000222975.1_ASM22297v1_genomic.fna.gz
mv GCF_000222975.1_ASM22297v1_genomic.fna.gz Ecoli.fasta.gz
gunzip Ecoli.fasta.gz
#Yersinia Pestis
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/Escherichia_coli/reference/GCF_000005845.2_ASM584v2/GCF_000005845.2_ASM584v2_genomic.fna.gz
mv GCF_000005845.2_ASM584v2_genomic.fna.gz Yerpes.fasta.gz
gunzip Yerpes.fasta.gz
nextflow run isugifNF/assemblyStats --genomes "*.fasta" --options "-l bacteria_odb10" -profile docker --threads 4
Expected output
N E X T F L O W ~ version 20.07.1
Launching `isugifNF/assemblyStats` [sad_boyd] - revision: a4272e736e [master]
executor > local (7)
[- ] process > runBUSCOlist -
[a1/5af149] process > runAssemblyStats (1) [100%] 2 of 2 ✔
[17/bb92c9] process > runAssemblathonStats (2) [100%] 2 of 2 ✔
[28/1103ba] process > setupBUSCO [100%] 1 of 1 ✔
[ba/962b47] process > runBUSCO (1) [100%] 2 of 2 ✔
Completed at: 12-Nov-2020 12:55:13
Duration : 1m 7s
CPU hours : (a few seconds)
Succeeded : 7
This workflow was built by Andrew Severin (@isugif) and Jennifer Chang (@j23414)
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ssh://[email protected]/isugifNF/assemblyStats.git: Auth fail
This occurs if you have not set up github authorization on your remote machine yet. See this Introduction to Github Tutorial on how to set up an ssh key.
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If you are planning on running this program more than once or more than one workflow it is best to set the NXF_SINGULARITY_CACHEDIR to a common location
export NXF_SINGULARITY_CACHEDIR=/location/of/singularity/container/folderPlace that in your
.bashrcfile. -
If it takes a really long time for your singularity images to be downloaded using nextflow, you can do it manually using
singularity pullthe first time.