├── .gitignore ├── LICENSE ├── README.md ├── data ├── MN996532.fasta ├── img │ ├── ani.png │ ├── dendrograms.png │ ├── gardtrack.png │ ├── gff.png │ ├── msa.png │ ├── ncbi.png │ ├── refinements.png │ └── seqs.png └── query_seqs │ ├── AY278741.fasta │ ├── DQ022305.fasta │ ├── FJ425189.fasta │ ├── KF367457.fasta │ ├── MG772933.fasta │ └── MN908947.fasta ├── docker └── Dockerfile ├── index.html ├── main.nf ├── nextflow.config └── templates ├── idplot.py └── jsontofasta.py /.gitignore: -------------------------------------------------------------------------------- 1 | work/ 2 | results/ 3 | .nextflow* 4 | -------------------------------------------------------------------------------- /LICENSE: -------------------------------------------------------------------------------- 1 | MIT License 2 | 3 | Copyright (c) 2020 Joe Brown 4 | 5 | Permission is hereby granted, free of charge, to any person obtaining a copy 6 | of this software and associated documentation files (the "Software"), to deal 7 | in the Software without restriction, including without limitation the rights 8 | to use, copy, modify, merge, publish, distribute, sublicense, and/or sell 9 | copies of the Software, and to permit persons to whom the Software is 10 | furnished to do so, subject to the following conditions: 11 | 12 | The above copyright notice and this permission notice shall be included in all 13 | copies or substantial portions of the Software. 14 | 15 | THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR 16 | IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, 17 | FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE 18 | AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER 19 | LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, 20 | OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE 21 | SOFTWARE. 22 | -------------------------------------------------------------------------------- /README.md: -------------------------------------------------------------------------------- 1 | Compare similar sequences (\*.fasta) to a reference (.fasta). 2 | 3 | See the example report at: https://brwnj.github.io/idplot/ 4 | 5 | # Setup 6 | 7 | Nextflow is used to run the pipeline. Its installation instructions 8 | can be found at https://www.nextflow.io/ or installed via [conda](https://docs.conda.io/projects/conda/en/latest/user-guide/install/) by 9 | way of [bioconda](https://bioconda.github.io/user/install.html). Bioconda 10 | includes a complete setup guide at https://bioconda.github.io/user/install.html. 11 | 12 | Once your install completes and your channels are configured, run: 13 | 14 | ``` 15 | conda install nextflow 16 | ``` 17 | 18 | # Usage 19 | 20 | Executing the workflow using nextflow: 21 | 22 | ``` 23 | nextflow run brwnj/idplot -latest -with-docker \ 24 | --reference data/MN996532.fasta \ 25 | --fasta 'data/query_seqs/*.fasta' 26 | ``` 27 | 28 | This generally takes only a few minutes to complete which enables rapid 29 | screening for localized sequence similarity. 30 | 31 | ## Parameters 32 | 33 | The reference sequence (`--reference`) should be a fasta with only one 34 | sequence in it. Query sequences (`--fasta`) may either be single sequence 35 | files or multi-sequence fasta files and you can specify more than one 36 | using wildcards ('\*'). 37 | 38 | Example sequences are found in `data/query_sequences`. 39 | 40 | By default, output is written to `./results/idplot.html` and can 41 | be opened with an internet browser. 42 | 43 | An example report is available at: https://brwnj.github.io/idplot/ 44 | 45 | ## Using a custom alignment 46 | 47 | In some cases it may be necessary to manually correct an alignment. In 48 | this case, `idplot` can accept the alignment and skip its internal 49 | alignment step. To do so, run: 50 | 51 | ``` 52 | nextflow run brwnj/idplot -latest -with-docker \ 53 | --alignment my_alignment_msa.fasta 54 | ``` 55 | 56 | The first sequence in the file will be used as the reference (root) 57 | sequence. 58 | 59 | Options `--reference` and `--fasta` are both omitted in this case. 60 | 61 | ## Including breakpoint detection 62 | 63 | We have opted to employ GARD via [HyPhy](https://github.com/veg/hyphy) to 64 | identify breakpoints. For each GARD fit iteration, we pull the sequences 65 | for each breakpoint and build a tree using [FastTree](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009490). 66 | 67 | An example command enabling GARD with 12 MPI processes: 68 | 69 | ``` 70 | nextflow run brwnj/idplot -latest -with-docker \ 71 | --reference data/MN996532.fasta \ 72 | --fasta 'data/query_seqs/*.fasta' \ 73 | --gard --cpus 12 74 | ``` 75 | 76 | ## Including a reference annotation 77 | 78 | Publicly available reference sequences, like in NCBI, often have an accompanying annotation 79 | that can be included within the ANI plot. On NCBI, one can get a GFF from the reference page under Send to: 80 | 81 | ![ani](data/img/ncbi.png) 82 | 83 | ``` 84 | nextflow run brwnj/idplot -latest -with-docker \ 85 | --reference data/MN996532.fasta \ 86 | --fasta 'data/query_seqs/*.fasta' \ 87 | --gff MN996532.gff3 88 | ``` 89 | 90 | Within the report, this renders as: 91 | 92 | ![gff](data/img/gff.png) 93 | 94 | As GFFs may have multiple feature types, we allow the reader to select their preferred feature type from the report header. 95 | 96 | Coordinates in the original GFF will likely not match what is being displayed. Start and end 97 | coordinates are updated based on gaps introduced into the reference sequence during multiple 98 | sequence alignment. 99 | 100 | # Interpreting the report 101 | 102 | ## Multiple sequence alignment 103 | 104 | ![msa](data/img/msa.png) 105 | 106 | The reference sequence is fully colored in. Hovering along the reference 107 | shows the base for a given color. 108 | 109 | Query sequences are colored at mismatches and gaps (gray). 110 | 111 | ## Percent ID (ANI) 112 | 113 | ![ani](data/img/ani.png) 114 | 115 | Percent ID is calculated across the window (default 500 bp) with the 116 | value being plotted at the center point. A 500 bp window will have 250 117 | bp dead spots at the beginning and end of the reference length. No 118 | special treatment is given with respect to sequence content. 119 | 120 | ## Sequences 121 | 122 | ![seqs](data/img/seqs.png) 123 | 124 | Sequence selection is based on the level of x-axis zoom of the plot. Sequence gaps can be removed using the toggle. The selected region can be copied to clipboard, sent directly to BLAST (when selection length is less than 8kb), or all sequences for a given region can be exported to FASTA. 125 | 126 | ## With GARD results 127 | 128 | Including `--gard` in your nextflow command adds breakpoint detection and updates available data and visualizations in the report. 129 | 130 | ### Breakpoints track 131 | 132 | ![gardtrack](data/img/gardtrack.png) 133 | 134 | Regions identified by GARD as breakpoints are highlighted between the 135 | MSA and ANI plots. Clicks on the regions will navigate to the respective dendrogram. 136 | 137 | ### Dendrograms 138 | 139 | ![dendrograms](data/img/dendrograms.png) 140 | 141 | Per region dendrograms are generated using [FastTree](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009490) based off of regions identified by GARD. 142 | 143 | Hovering over regions highlights the respective region in the GARD breakpoints track. 144 | 145 | Clicking the region link will zoom the plot to facilitate downloading the sequence content for a given region. 146 | 147 | ### Refinements 148 | 149 | ![refinements](data/img/refinements.png) 150 | 151 | Breakpoints are identified over iterations by GARD, often to an unhelpful degree. This plot allows the user to explore breakpoints and trees across all GARD iterations. Selecting a new point will update the dendrograms and GARD breakpoints track. 152 | -------------------------------------------------------------------------------- /data/MN996532.fasta: -------------------------------------------------------------------------------- 1 | >MN996532 2 | 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3 | -------------------------------------------------------------------------------- /data/query_seqs/DQ022305.fasta: -------------------------------------------------------------------------------- 1 | >DQ022305 2 | 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3 | -------------------------------------------------------------------------------- /data/query_seqs/FJ425189.fasta: -------------------------------------------------------------------------------- 1 | >FJ425189 2 | 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3 | -------------------------------------------------------------------------------- /data/query_seqs/KF367457.fasta: -------------------------------------------------------------------------------- 1 | >KF367457 2 | 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3 | -------------------------------------------------------------------------------- /data/query_seqs/MG772933.fasta: -------------------------------------------------------------------------------- 1 | >MG772933 2 | 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3 | -------------------------------------------------------------------------------- /data/query_seqs/MN908947.fasta: -------------------------------------------------------------------------------- 1 | >MN908947 2 | 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3 | -------------------------------------------------------------------------------- /docker/Dockerfile: -------------------------------------------------------------------------------- 1 | FROM continuumio/miniconda3:4.8.2 2 | 3 | LABEL version="1.1.3" maintainer="Joe Brown " 4 | 5 | RUN apt-get update && apt-get install -y g++ make procps 6 | RUN conda create -n main -c bioconda -c conda-forge python==3.8.3 mafft==7.471 hyphy==2.5.15 fasttree==2.1.10 7 | RUN echo "source activate main" > ~/.bashrc 8 | ENV PATH /opt/conda/envs/main/bin:$PATH 9 | -------------------------------------------------------------------------------- /main.nf: -------------------------------------------------------------------------------- 1 | params.help = false 2 | if (params.help) { 3 | log.info """ 4 | ----------------------------------------------------------------------- 5 | 6 | idplot: compare similar sequences to a reference 7 | ================================================ 8 | 9 | Output is written to /idplot.html and can 10 | be opened with your internet browser. 11 | 12 | required 13 | -------- 14 | --reference Fasta sequence that will act as the root sequence. 15 | --fasta One ('my.fasta') or multiple ('*.fasta') query sequences 16 | to compare to `--reference`. 17 | 18 | OR 19 | 20 | --alignment Pre-aligned sequences where the first sequence is your root. 21 | 22 | options 23 | ------- 24 | --outdir Base results directory for output. 25 | Default: '/.results' 26 | --window The sliding window size across the reference genome upon 27 | which to calculate similarity. 28 | Default: 500 29 | --gard Run GARD for breakpoint detection. 30 | Default: false 31 | --gff Reference annotation file in .gff or .gff3 format. 32 | Coordinates will be adjusted to include gaps introduced 33 | during sequence alignment. 34 | Default: false 35 | --cpus Threads for multi-threaded processes. 36 | Default: 1 37 | ----------------------------------------------------------------------- 38 | """.stripIndent() 39 | exit 0 40 | } 41 | // required arguments 42 | params.reference = false 43 | params.fasta = false 44 | params.alignment = false 45 | if( params.alignment ) { 46 | Channel 47 | .fromPath(params.alignment, checkIfExists: true) 48 | .into { alignment_gard_ch; alignment_json_ch; alignment_report_ch } 49 | mafft_ch = Channel.empty() 50 | reference = false 51 | } else { 52 | if( !params.reference ) { exit 1, "Neither --reference NOR --alignment are defined" } 53 | reference = file(params.reference) 54 | if( !reference.exists() ) { exit 1, "Reference [${reference}] does not exist." } 55 | if( !params.fasta ) { exit 1, "--fasta is not defined" } 56 | Channel 57 | .fromPath(params.fasta, checkIfExists: true) 58 | .set { mafft_ch } 59 | } 60 | 61 | gff = params.gff ? file(params.gff) : false 62 | 63 | process mafft { 64 | publishDir path: "${params.outdir}/", mode: "copy" 65 | cpus params.cpus.toInteger() 66 | 67 | input: 68 | path(reference) 69 | path(query) from mafft_ch.collect() 70 | 71 | output: 72 | path("${reference.baseName}.msa.fasta") into (msa_gard_ch, msa_json_ch, msa_report_ch) 73 | 74 | when: 75 | params.reference 76 | 77 | script: 78 | """ 79 | cat ${reference} > mafft_input.fasta 80 | cat ${query} >> mafft_input.fasta 81 | mafft --auto --thread ${task.cpus} --maxiterate 1000 --globalpair mafft_input.fasta > ${reference.baseName}.msa.fasta 82 | """ 83 | } 84 | 85 | msa_gard_input_ch = (params.reference ? msa_gard_ch : alignment_gard_ch) 86 | msa_json_input_ch = (params.reference ? msa_json_ch : alignment_json_ch) 87 | msa_report_input_ch = (params.reference ? msa_report_ch : alignment_report_ch) 88 | 89 | process gard { 90 | publishDir path: "${params.outdir}/gard/", mode: "copy" 91 | tag "${msa}" 92 | cpus params.cpus.toInteger() 93 | 94 | input: 95 | path(msa) from msa_gard_input_ch 96 | 97 | output: 98 | path("${msa.baseName}.json") into (gard_output_ch, gard_output_secondary_ch) 99 | 100 | when: 101 | params.gard 102 | 103 | script: 104 | cmd = params.nompi ? "hyphy" : "mpirun -np ${task.cpus} --allow-run-as-root --oversubscribe HYPHYMPI" 105 | // rv: None, GDD, Gamma 106 | """ 107 | $cmd CPU=${task.cpus} gard --type nucleotide --code Universal \ 108 | --alignment ${msa} --rv GDD --model JTT --rate-classes 4 --output ${msa.baseName}.json 109 | """ 110 | } 111 | 112 | gard_json_ch = (params.gard ? gard_output_ch : [""]) 113 | gard_report_ch = (params.gard ? gard_output_secondary_ch : [""]) 114 | 115 | process jsontofasta { 116 | input: 117 | path(msa) from msa_json_input_ch 118 | file(json) from gard_json_ch 119 | 120 | output: 121 | path("*.fa") into selection_ch 122 | 123 | when: 124 | params.gard 125 | 126 | script: 127 | template "jsontofasta.py" 128 | } 129 | 130 | 131 | process fasttree { 132 | input: 133 | path(fasta) from selection_ch.flatten() 134 | 135 | output: 136 | path("${fasta.simpleName}.tree") into tree_output_ch 137 | 138 | script: 139 | """ 140 | fasttree -nt -gamma -spr 4 -quiet ${fasta} > ${fasta.simpleName}.tree 141 | """ 142 | } 143 | 144 | tree_report_ch = (params.gard ? tree_output_ch : [""]) 145 | 146 | process idplot { 147 | publishDir path: "${params.outdir}/", mode: "copy" 148 | 149 | input: 150 | path(msa) from msa_report_input_ch 151 | file(json) from gard_report_ch 152 | file(trees) from tree_report_ch.collect() 153 | file(gff) 154 | 155 | output: 156 | path("idplot.html") 157 | 158 | script: 159 | template "idplot.py" 160 | } 161 | -------------------------------------------------------------------------------- /nextflow.config: -------------------------------------------------------------------------------- 1 | // Configurable variables 2 | params { 3 | // the genomic window size for calculating percent ID 4 | window = 500 5 | // where to place the results 6 | outdir = './results' 7 | gard = false 8 | gff = false 9 | nompi = false 10 | cpus = 1 11 | } 12 | 13 | process { 14 | time = 72.h 15 | memory = 4.GB 16 | cpus = 1 17 | container = 'brwnj/idplot:v1.1.3' 18 | cache = 'deep' 19 | errorStrategy = 'finish' 20 | withName: gard { 21 | time = 336.h 22 | memory = { 16.GB * task.attempt } 23 | } 24 | } 25 | 26 | process.shell = ['/bin/bash', '-euo', 'pipefail'] 27 | 28 | timeline { 29 | enabled = true 30 | file = "${params.outdir}/logs/timeline.html" 31 | } 32 | report { 33 | enabled = true 34 | file = "${params.outdir}/logs/report.html" 35 | } 36 | trace { 37 | enabled = true 38 | file = "${params.outdir}/logs/trace.txt" 39 | } 40 | 41 | manifest { 42 | name = 'brwnj/idplot' 43 | author = 'Joe Brown' 44 | description = "idplot: compare similar sequences to a reference" 45 | version = '1.1.1' 46 | nextflowVersion = '>=0.32.0' 47 | homePage = 'https://github.com/brwnj/idplot' 48 | mainScript = 'main.nf' 49 | } 50 | -------------------------------------------------------------------------------- /templates/jsontofasta.py: -------------------------------------------------------------------------------- 1 | #!/usr/bin/env python 2 | 3 | import json 4 | from itertools import groupby 5 | 6 | 7 | json_file = "$json" 8 | alignments = "$msa" 9 | # standardized workflow file name 10 | output_prefix = alignments.rpartition(".msa.fasta")[0] 11 | regions = set() 12 | 13 | 14 | def read_fasta(fh): 15 | for header, group in groupby(fh, lambda line: line[0] == ">"): 16 | if header: 17 | line = next(group) 18 | name = line.strip() 19 | else: 20 | seq = "".join(line.strip() for line in group) 21 | yield name, seq 22 | 23 | 24 | with open(json_file) as fh: 25 | gard = json.load(fh) 26 | total_len = int(gard["input"]["number of sites"]) 27 | 28 | for i in range(len(gard["improvements"])): 29 | start = 0 30 | for bp in gard["improvements"][f"{i}"]["breakpoints"]: 31 | end = bp[0] 32 | regions.add(f"{start}_{end}") 33 | start = int(end) + 1 34 | regions.add(f"{start}_{total_len}") 35 | 36 | # grab the sequences 37 | seqs = dict() 38 | with open(alignments) as fh: 39 | for name, seq in read_fasta(fh): 40 | seqs[name] = seq.replace("u", "t").replace("U", "T") 41 | 42 | for region in regions: 43 | filename = f"{output_prefix}_{region}.fa" 44 | start, end = region.split("_") 45 | start = int(start) 46 | end = int(end) 47 | with open(filename, "w") as fh: 48 | for name, seq in seqs.items(): 49 | print(name, seq[start:end+1], sep="\\n", file=fh) 50 | --------------------------------------------------------------------------------