pipeline-umi-amplicon is a pipeline for generating high accuracy single molecule reads using unique molecular identifiers (UMIs) from amplicon data. The pipeline accepts FASTQ-format sequence files as input and outputs both aligned reads and QC stats.

The pipeline performs the following steps:

• Reads are mapped to reference genome using minimap2
• Separate into amplicons
• Extract UMI sequences for all reads
• Cluster UMI sequences per amplicon using vsearch and compute high accuracy consensus reads
• Align high accuracy conesnsus reads and perform simple variant calling (optional)

The following software packages must be installed prior to running:

• miniconda3 - please refer to installation instructions.

After installing miniconda3, install the pipeline as follows:

# Get pipeline
# git clone https://github.com/nanoporetech/pipeline-umi-amplicon.git 
git clone https://github.com/SemiQuant/pipeline-umi-amplicon.git
# Change to directory
cd pipeline-umi-amplicon
# Create conda environment with all dependencies
conda env create -f environment.yml
# Activate environment
conda activate pipeline-umi-amplicon
# Install python packages provided by pipeline-umi-amplicon
cd lib && pip install . && cd ..

# To test if the installation was successful run
snakemake -j 1 -pr --configfile config.yml
# Deactivate environment
conda deactivate

To run the pipeline the following input files are required:

Tab separated and needs a unique name:

chr1    107167322       107168239       target_a_chr1_107167756_T_C

The main output files created by the pipeline are:

After the a pipeline analysis has completed, the aligned reads can be found at {output_folder}/{run_name}/align/{amplicon_name}_final.bam e.g. example_egfr_single_read_run/align/EGFR_917_final.bam.

To run the pipeline with default settings invoke snakemake as follows.

$ snakemake -j 30 reads --configfile config.yml

-j specifies how many CPU cores will be used by the pipeline. reads is the default target (see Targets); this will run all steps required to produce aligned high accuracy consensus reads. Please see the example config files for the required parameters.

The pipeline accepts several input parameters. They can either be changed in the config.yml file or specified when running snakemake.

For example:

snakemake -j 30 reads --config input_fastq=data reference_fasta=data/example_egfr_reference.fasta targets_bed=data/example_egfr_amplicon.bed

These parameters have to be specified to run the pipeline.

See config.yml

(c) 2020 Oxford Nanopore Technologies Ltd.

This Source Code Form is subject to the terms of the Mozilla Public License, v. 2.0. If a copy of the MPL was not distributed with this file, You can obtain one at http://mozilla.org/MPL/2.0/.

If you use this pipeline please cite:

• Li, H. (2018). Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics, 34(18), 3094–3100. https://doi.org/10.1093/bioinformatics/bty191
• Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, and 1000 Genome Project Data Processing Subgroup, The Sequence alignment/map (SAM) format and SAMtools, Bioinformatics (2009) 25(16) 2078-9 [19505943]
• Rognes T, Flouri T, Nichols B, Quince C, Mahé F. (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584. doi: 10.7717/peerj.2584
• For optional variant calling: Koboldt, D., Zhang, Q., Larson, D., Shen, D., McLellan, M., Lin, L., Miller, C., Mardis, E., Ding, L., & Wilson, R. (2012). VarScan 2: Somatic mutation and copy number alteration discovery in cancer by exome sequencing Genome Research DOI: 10.1101/gr.129684.111

Research releases are provided as technology demonstrators to provide early access to features or stimulate Community development of tools. Support for this software will be minimal and is only provided directly by the developers. Feature requests, improvements, and discussions are welcome and can be implemented by forking and pull requests. However much as we would like to rectify every issue and piece of feedback users may have, the developers may have limited resource for support of this software. Research releases may be unstable and subject to rapid iteration by Oxford Nanopore Technologies.

• Number of reads collapsed in the header of the bam file
• Check that balance reads is working as its meant to
• Add handling of errors if no reads aligned to a reference
• Add stats output and plots, number of umis, umi distribution, snps plot umi vs raw, read alignment stats

Done

• I added a fastq filter options (min read length and average quality) using filtlong
• The user can also now pass options to the variant calling using the config file
• It runs as before when in defaults mode