This pipeline extracts paired gene and barcode sequences from long-read sequencing data produced from gene shuffling/protein engineering experiments in the Plesa Lab at the University of Oregon Knight Campus. It is designed to work with data from both Oxford Nanopore and PacBio sequencing platforms.
The expected input format depends on the sequencing platform.
For PacBio data:
- 1 FASTQ file containing CCS reads (*.ccs.fastq)
For Oxford Nanopore data:
- Multiple FASTQ files containing demultiplexed sequencing data. (Tested on a set of 5 files)
In addition to the sequencing data, the pipeline requires files containing other sequences present in the DNA constructs:
- 1 FASTA file containing 3 conserved regions
- with header lines "CR1", "CR2", "CR3"
- CR1 sequence should end with the ATG start codon.
- 1 FASTA file containing barcode sequences for demultiplexing, with barcode number indicated in the header lines.
The primary output of the pipeline is a .csv file containing paired genes and barcodes. The columns contain:
- Barcode sequence
- Barcode count
- Consensus gene sequence
- Amino acid translation (to first stop codon)
- Amino acid translation (complete sequence)
Intermediate files are produced by each step of the pipeline. More details about outputs from each step are in /src/README.md.
--platform: String specifying the sequencing platform origin of the data. Only accepts pacbio or nanopore. Defaults to pacbio.
--infile: Path to the raw FASTQ files. If sending multiple FASTQs at once (as for Oxford Nanopore data), use pattern matching to capture files. (e.g. /folder/data/nanopore/barcode0*d_test.fastq)
--outdir: Output directory for all results files. Will be populated with subfolders containing outputs for each process step. Default is <current working directory>/results/<run start timestamp>-results
--crfile: Path to FASTA file containing conserved region sequences.
--indexfile: Path to FASTA file containing library indices.
--length: Maximum monomer length in nt. Default is 1200. Sequences longer than length will not be included in the analysis.
--help: Prints help information.
The location of starcode will need to be updated in the beginning of the script to reflect where it is installed in your local environment.
| Tool | Version |
|---|---|
| Nextflow | 22.10.3 |
| Java | 11 (up to 18) |
| Bash | 3.2 or later |
| Python | 3.10 |
| matplotlib | 3.7.1 |
| regex | 2022.10.31 |
| Lima | 2.7.1 |
| Biopython | 1.81 |
| bioawk | 1.0 |
| LAST | 1452 |
| lamassemble | |
| starcode | 1.4 |
Starcode
Filion, Guillaume. Starcode: Sequence clustering based on all-pairs search. Latest release: 02 Nov 2020. https://github.com/gui11aume/starcode
LAST
Frith, Martin. last. Latest update: 17 Oct 2022. https://gitlab.com/mcfrith/last
Nextflow
Seqera Labs. Nextflow. Latest release: 27 Oct 2022. https://github.com/nextflow-io/nextflow
lamassemble
M. C. Frith, S. Mitsuhashi, K. Katoh, lamassemble: Multiple Alignment and Consensus Sequence of Long Reads. Methods Mol Biol 2231, 135-145 (2021).
S. M. Karst, R. M. Ziels, R. H. Kirkegaard, E. A. Sørensen, D. McDonald, Q. Zhu, R. Knight, M. Albertsen, High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing. Nat Methods 18, 165–169 (2021).
Figures presented at Genomics in Action 2023 were created with Biorender. https://biorender.com/