### Install VarVamp for Development Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/installation.md Clone the VarVamp repository and install it locally in editable mode for development purposes. ```shell git clone https://github.com/jonas-fuchs/varVAMP cd varVAMP pip install . varvamp -v ``` -------------------------------- ### Install VarVamp using PyPI Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/installation.md Use this command to install the latest stable version of VarVamp from the Python Package Index. ```shell pip install varvamp ``` -------------------------------- ### Verify VarVamp Installation Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/installation.md Run this command after installation to confirm that VarVamp is correctly installed and accessible in your environment. ```shell varvamp -v ``` -------------------------------- ### Install BLAST on Linux Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/preparing_the_data.md Installs the BLAST+ package on a Red Hat Linux system using an RPM file. This is a prerequisite for using VarVAMP's primer BLAST feature. ```shell rpm -ivh ncbi-blast-2.2.18-1.x86_64.rpm ``` -------------------------------- ### Install VarVamp using Conda Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/installation.md Create a dedicated Conda environment for VarVamp to manage dependencies and activate it for use. ```shell conda create -n varvamp varvamp conda activate varvamp ``` -------------------------------- ### Running VarVAMP with Custom Config (Windows) Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/usage.md This Windows command prompt example demonstrates setting the VARVAMP_CONFIG environment variable to 'custom_config.py' before running the varvamp command. This applies the custom configuration. ```shell set "VARVAMP_CONFIG=custom_config.py" varvamp qpcr input_alignment.fasta my_results ``` -------------------------------- ### SuperScript-IV RT-PCR Master Mix Reaction Setup Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/wet_lab_protocol.md Reagents and volumes for setting up the PCR reaction for amplicon generation. ```text 12,5 μL 2X Platinum™ SuperFi™ RT-PCR Master Mix 0.25 μL SuperScript™ IV RT Mix 0.75 µl Nuclease-free water 9.00 µl Template RNA (0.01 pg to 1 μg total RNA) 1.25 μl varVAMP´s forward primer (10 μM) 1.25 μl varVAMP´s reverse primer (10 μM) Total volume: 25 µl ``` -------------------------------- ### Cluster Sequences with vsearch Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/preparing_the_data.md Clusters sequences based on sequence identity using the vsearch tool. This is recommended for highly diverse datasets to improve VarVAMP's performance. A sequence identity between 0.8 and 0.85 is suggested as a starting point. ```shell vsearch --cluster_fast --clusters --id ``` -------------------------------- ### Full VarVAMP Usage and Arguments Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/usage.md Provides a comprehensive overview of VarVAMP's command-line structure, including positional and optional arguments, and available modes. ```shell usage: varvamp --help varvamp [mode optional arguments] ``` ```shell positional arguments: input alignment file and dir to write results optional arguments: -h, --help show this help message and exit --verbose, --no-verbose show varvamp console output (default: True) -v, --version show program's version number and exit varvamp mode: {single,tiled,qpcr} single design primers for single amplicons tiled design primers for whole genome sequencing qpcr design qPCR primers ``` -------------------------------- ### Minimal VarVAMP Usage Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/usage.md The most basic way to run VarVAMP requires specifying the mode, alignment file, and output directory. ```shell varvamp ``` -------------------------------- ### Default Primer Size Configuration Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/usage.md This snippet shows the default configuration for primer sizes in VarVAMP's default config file. It defines the minimum, maximum, and optimal primer sizes. ```python PRIMER_SIZES = (18, 24, 21) # size (min, max, opt) ``` -------------------------------- ### Custom Primer Size Configuration Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/usage.md This snippet demonstrates how to create a custom configuration file to change the optimal primer size from 21 to 22 nucleotides. Comments are ignored by VarVAMP. ```python PRIMER_SIZES = (18, 24, 22) # size (min, max, opt); changed opt from 21 to prefer somewhat longer primers ``` -------------------------------- ### Running VarVAMP with Custom Config in a Path with Spaces (Linux) Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/usage.md This Linux shell command illustrates running VarVAMP with a custom configuration file located in a directory with spaces in its name. Quotes are used to handle the path correctly. ```shell VARVAMP_CONFIG="/home/me/my varvamp configs/sars-cov-2_config.py" varvamp tiled ncov_alignment.fasta my_results ``` -------------------------------- ### VarVAMP Tiled Mode Usage Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/usage.md Details the command-line usage for the 'tiled' mode, designed for primer design in whole genome sequencing. Includes specific optional arguments. ```shell usage: varvamp tiled [optional arguments] ``` ```shell optional arguments: -h, --help show this help message and exit -t, --threshold consensus threshold - if not set it will be automatically determined (higher values result in higher specificity at the expense of found primers) -a , --n-ambig max number of ambiguous characters in a primer -db None, --database None location of the BLAST db -th 1, --threads 1 number of threads for BLAST search and deltaG calculations --name varVAMP name of the scheme -ol 1000, --opt-length 1000 optimal length of the amplicons -ml 1500, --max-length 1500 max length of the amplicons -o 100, --overlap 100 min overlap of the amplicon inserts ``` -------------------------------- ### Run VarVAMP with Primer BLAST Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/preparing_the_data.md Executes VarVAMP with the primer BLAST feature, specifying a pre-created BLAST database. Ensure the `-th` argument is set for the number of threads used in the BLAST search. ```shell varVAMP tiled -db off_targets/off_targets alignment.fa varvamp_results ``` -------------------------------- ### Create BLAST Database Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/preparing_the_data.md Creates a BLAST nucleotide database from a multi-FASTA file containing potential off-target sequences. This database is used by VarVAMP for primer analysis. ```shell makeblastdb -in potential_off_targets.fasta -out off_targets -dbtype 'nucl' -hash_index ``` -------------------------------- ### VarVAMP Single Mode Usage Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/usage.md Details the command-line usage for the 'single' mode, which is used for designing primers for single amplicons. Includes specific optional arguments. ```shell usage: varvamp single [optional arguments] ``` ```shell optional arguments: -h, --help show this help message and exit -t, --threshold consensus threshold - if not set it will be automatically determined (higher values result in higher specificity at the expense of found primers) -a , --n-ambig max number of ambiguous characters in a primer -db None, --database None location of the BLAST db -th 1, --threads 1 number of threads for BLAST search and deltaG calculations --name varVAMP name of the scheme -ol 1000, --opt-length 1000 optimal length of the amplicons -ml 1500, --max-length 1500 max length of the amplicons -n inf, --report-n inf report the top n best hits ``` -------------------------------- ### VarVAMP qPCR Mode Usage Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/usage.md Details the command-line usage for the 'qpcr' mode, used for designing qPCR primers. Includes specific optional arguments related to primer and probe design, and secondary structure testing. ```shell usage: varvamp qpcr [optional arguments] ``` ```shell optional arguments: -h, --help show this help message and exit -t, --threshold threshold for consensus nucleotides - increase for higher specificity at the expense of found primers -a , --n-ambig max number of ambiguous characters in a primer -db None, --database None location of the BLAST db -th 1, --threads 1 number of threads for BLAST search and deltaG calculations --name varVAMP name of the scheme -pa , --pn-ambig max number of ambiguous characters in a probe -n 50, --test-n 50 test the top n qPCR amplicons for secondary structures at the minimal primer temperature -d -3, --deltaG -3 minimum free energy (kcal/mol/K) cutoff at the lowest primer melting temperature ``` -------------------------------- ### Running VarVAMP with Custom Config (Linux) Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/usage.md This Linux shell command shows how to run VarVAMP with a custom configuration file named 'custom_config.py' located in the current directory. The VARVAMP_CONFIG variable points to the custom file. ```shell VARVAMP_CONFIG=custom_config.py varvamp qpcr input_alignment.fasta my_results ``` -------------------------------- ### Concatenate FASTA Files Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/preparing_the_data.md Combines all FASTA files in the current directory into a single multi-FASTA file. This is the first step in preparing your sequence data. ```shell cat *.fasta > my_sequences.fasta ``` -------------------------------- ### Pull VarVamp Docker Image Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/installation.md Download the VarVamp Docker image from Quay.io. Replace with the appropriate version tag. ```shell docker pull quay.io/biocontainers/varvamp: ``` -------------------------------- ### Align Sequences with MAFFT Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/preparing_the_data.md Aligns sequences in a multi-FASTA file using the MAFFT tool. This is a crucial step for preparing phylogenetically meaningful input for VarVAMP. ```shell mafft my_sequences.fasta > my_alignment.fasta ``` -------------------------------- ### RNA Isolation Reagents Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/wet_lab_protocol.md List of reagents and volumes required for RNA isolation using the QIAmp Viral RNA Kit. ```text 560 µl prepared Buffer AVL (containing 5.6 µg carrier RNA) 140 µl cell-culture supernatant Incubate 10 min 560 µl ethanol (100%) 500 µl Buffer AW1 500 µl Buffer AW2 Elute in 60 µl Buffer AVE ``` -------------------------------- ### Primer Permutation Penalty Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/how_varvamp_works.md Penalizes primers based on the number of their permutations. Set to 0 to ignore permutation counts. ```python3 PRIMER_PERMUTATION_PENALTY ``` -------------------------------- ### Touchdown PCR Cycler Program Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/wet_lab_protocol.md The touchdown PCR program parameters including temperatures, durations, and cycles. ```text 55°C | 60 min | 1 98°C | 2 min | 1 98°C | 10s | 10 63°C (-0.5°C/cycle) | 10s | 10 72°C | 2 min | 10 98°C | 10s | 35 58°C | 10s | 35 72°C | 2 min | 35 72°C | 5 min | 1 4°C | ∞ | 1 ``` -------------------------------- ### Primer Max Base Penalty Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/how_varvamp_works.md Excludes primers with base penalties exceeding this threshold. Penalties are based on deviations in size, GC content, and temperature. ```python PRIMER_MAX_BASE_PENALTY ``` -------------------------------- ### Primer 3 Penalty Source: https://github.com/jonas-fuchs/varvamp/blob/master/docs/how_varvamp_works.md Penalizes mismatches at the 3' end of primers. Adjust to favor amplicons with better 3' matching. Set to 0 to ignore 3' mismatches. ```python PRIMER_3_PENALTY ``` === COMPLETE CONTENT === This response contains all available snippets from this library. No additional content exists. Do not make further requests.