Pronounced: Scarf
Sc2rf can search genome sequences of SARS-CoV-2 for potential recombinants - new virus lineages that have (partial) genes from more than one parent lineage.
This is a very young project, started on March 5th, 2022. As such, proceed with care. Results may be wrong or misleading, and with every update, anything can still change a lot.
Anyway, I'm happy that scientists are already seeing benefits from Sc2rf and using it to prepare lineage proposals for cov-lineages/pango-designation.
Though I already have a lot of ideas and plans for Sc2rf (see at the bottom of this document), I'm very open for suggestions and feature requests. Please write an issue, start a discussion or get in touch via mail or twitter!
You need at least Python 3.6 and you need to install the requirements first. You might use something like python3 -m pip install -r requirements.txt to do that. There's a setup.py which you should probably ignore, since it's work in progress and does not work as intented yet.
Also, you need a terminal which supports ANSI control sequences to display colored text. On Linux, MacOS, etc. it should probably work.
On Windows, color support is tricky. On a recent version of Windows 10, it should work, but if it doesn't, install Windows Terminal from GitHub or Microsoft Store and run it from there.
Start with a .fasta file with one or more sequences which might contain recombinants. Your sequences have to be aligned to the reference.fasta. If they are not, you will get an error message like:
Sequence hCoV-19/Phantasialand/EFWEFWD not properly aligned, length is 29718 instead of 29903.
(For historical reasons, I always used Nextclade to get aligned sequences, but you might also use Nextalign or any other tool. Installing them is easy on Linux or MacOS, but not on Windows. You can also use a web-based tool like MAFFT.)
Then call:
sc2rf.py <your_filename.fasta>
If you just need some fasta files for testing, you can search the pango-lineage proposals for recombinant issues with fasta-files, or take some files from my shared-sequences repository, which might not contain any actual recombinants, but hundreds of sequences that look like they were!
By default, a lot filters are active to show only the likely recombinants, so that you can input 10000s of sequences and just get output for the interesting ones. If you want, you can disable all filters like that, which is only recommended for small input files with less than 100 sequences:
sc2rf.py --parents 1-35 --breakpoints 0-100 \
--unique 1 --max-ambiguous 10000 <your_filename.fasta>
or even
sc2rf.py --parents 1-35 --breakpoints 0-100 \
--unique 1 --max-ambiguous 10000 --force-all-parents \
--clades all <your_filename.fasta>
The meaning of these parameters is described below.
You can execute sc2rf.py -h to get excactly this help message:
usage: sc2rf.py [-h] [--primers [PRIMER ...]]
[--primer-intervals [INTERVAL ...]]
[--parents INTERVAL] [--breakpoints INTERVAL]
[--clades [CLADES ...]] [--unique NUM]
[--max-intermission-length NUM]
[--max-intermission-count NUM]
[--max-name-length NUM] [--max-ambiguous NUM]
[--force-all-parents]
[--select-sequences INTERVAL]
[--enable-deletions] [--show-private-mutations]
[--rebuild-examples] [--mutation-threshold NUM]
[--add-spaces [NUM]] [--sort-by-id [NUM]]
[--verbose] [--ansi] [--hide-progress]
[--csvfile CSVFILE]
[input ...]
Analyse SARS-CoV-2 sequences for potential, unknown recombinant
variants.
positional arguments:
input input sequence(s) to test, as aligned
.fasta file(s) (default: None)
optional arguments:
-h, --help show this help message and exit
--primers [PRIMER ...]
Filenames of primer set(s) to visualize.
The .bed formats for ARTIC and EasySeq
are recognized and supported. (default:
None)
--primer-intervals [INTERVAL ...]
Coordinate intervals in which to
visualize primers. (default: None)
--parents INTERVAL, -p INTERVAL
Allowed number of potential parents of a
recombinant. (default: 2-4)
--breakpoints INTERVAL, -b INTERVAL
Allowed number of breakpoints in a
recombinant. (default: 1-4)
--clades [CLADES ...], -c [CLADES ...]
List of variants which are considered as
potential parents. Use Nextstrain clades
(like "21B"), or Pango Lineages (like
"B.1.617.1") or both. Also accepts "all".
(default: ['20I', '20H', '20J', '21I',
'21J', 'BA.1', 'BA.2', 'BA.3'])
--unique NUM, -u NUM Minimum of substitutions in a sample
which are unique to a potential parent
clade, so that the clade will be
considered. (default: 2)
--max-intermission-length NUM, -l NUM
The maximum length of an intermission in
consecutive substitutions. Intermissions
are stretches to be ignored when counting
breakpoints. (default: 2)
--max-intermission-count NUM, -i NUM
The maximum number of intermissions which
will be ignored. Surplus intermissions
count towards the number of breakpoints.
(default: 8)
--max-name-length NUM, -n NUM
Only show up to NUM characters of sample
names. (default: 30)
--max-ambiguous NUM, -a NUM
Maximum number of ambiguous nucs in a
sample before it gets ignored. (default:
50)
--force-all-parents, -f
Force to consider all clades as potential
parents for all sequences. Only useful
for debugging.
--select-sequences INTERVAL, -s INTERVAL
Use only a specific range of input
sequences. DOES NOT YET WORK WITH
MULTIPLE INPUT FILES. (default: 0-999999)
--enable-deletions, -d
Include deletions in lineage comparision.
--show-private-mutations
Display mutations which are not in any of
the potential parental clades.
--rebuild-examples, -r
Rebuild the mutations in examples by
querying cov-spectrum.org.
--mutation-threshold NUM, -t NUM
Consider mutations with a prevalence of
at least NUM as mandatory for a clade
(range 0.05 - 1.0, default: 0.75).
--add-spaces [NUM] Add spaces between every N colums, which
makes it easier to keep your eye at a
fixed place. (default without flag: 0,
default with flag: 5)
--sort-by-id [NUM] Sort the input sequences by the ID. If
you provide NUM, only the first NUM
characters are considered. Useful if this
correlates with meaning full meta
information, e.g. the sequencing lab.
(default without flag: 0, default with
flag: 999)
--verbose, -v Print some more information, mostly
useful for debugging.
--ansi Use only ASCII characters to be
compatible with ansilove.
--hide-progress Don't show progress bars during long
task.
--csvfile CSVFILE Path to write results in CSV format.
(default: None)
An Interval can be a single number ("3"), a closed interval
("2-5" ) or an open one ("4-" or "-7"). The limits are inclusive.
Only positive numbers are supported.
To be written...
There already is a short Twitter thread which explains the basics.
virus_properties.jsoncontains data from LAPIS / cov-spectrum which uses data from NCBI GenBank, prepared and hosted by Nextstrain, see blog post.reference.fastais taken from Nextstrain's nextclade_data, see NCBI for attribution.mapping.csvis a modified version of the table on the covariants homepage by Nextstrain.- Example output / screenshot based on Sequences published by the German Robert-Koch-Institut.
- Primers:
- ARTIC primers CC-BY-4.0 by the ARTICnetwork project
EasySeq primers by Coolen, J. P., Wolters, F., Tostmann, A., van Groningen, L. F., Bleeker-Rovers, C. P., Tan, E. C., ... & Melchers, W. J.Removed until I understand the format if the.bedfile. There will be an issue soon.- midnight primers CC-BY-4.0 by Silander, Olin K, Massey University
The initial version of this program was written in cooperation with @flauschzelle.
- Move these TODOs into actual issues
- add disclaimer and link to pango-designation
- provide a sample file (maybe both
.fastaand.csv, as long as the csv step is still needed) - accept aligned fasta
- as input file
- as piped stream
- If we still accept csv/ssv input, autodetect the delimiter either by file name or by analysing the first line
- find a way to handle already designated recombinant lineages
- Output structured results
- csv
- html?
- fasta of all sequences that match the criteria, which enables efficient multi-pass strategies
- filter sequences
- by ID
- by metadata
- take metadata csv
- document the output in README
- check / fix
--enabled-deletions - adjustable threshold for mutation prevalence
- new color mode (with background color and monochrome text on top)
- new bar mode (with colored lines beneath each sequence, one for each example sequence, and "intermissions" shown in the color of the "surrounding" lineage, but not as bright)
- interactive mode, for filtering, reordering, etc.
- sort sequences within each block
- re-think this whole "intermission" concept
- select a single sequence and let the tool refine the choice of parental sequences, not just focusing on commonly known lineages (going up and down in the tree)
- use more common terms to describe things (needs feedback from people with actual experience in the field)