Background

This repository’s code and data accompany the manuscript, Uncovering Position-Specific Patterns in Codon and Codon-Pair Usage in Model Genes. Current strategies for optimizing gene therapeutics and recombinant protein production typically rely on universal host codon usage indices. However, it is becoming increasingly common to consider specific traits of the gene. In this study, we investigate position-specific variations in codon and adjacent codon-pair usage biases, offering potential for more tailored gene engineering approaches to optimize translational kinetics and the folding of encoded products.

We focus our analysis on the coding sequences of four coagulation factors: ADAMTS13, von Willebrand factor, Factor VIII, and Factor IX, which have been used in therapeutic applications. By aligning transcript homologs with human sequences for each gene using Discontiguous Megablast and MACSE, we assess “sequence-position-specific” codon and codon-pair usage biases. Species with homologs ranged from Primates and Artiodactyla (Even-toed Ungulates) to Testudines.

Position-specific positive codon-pair usage biases were observed that contrasted with codon-pair usage biases derived with conventional methods. Moreover, we observed that codon and codon-pair usages are highly associated at sequence positions despite little or no association in an alignment universally. Statistical significance of position-specific codon bias was quantified with permutation tests modified for codon alignments. The distinct biases observed at different positions in coding sequences highlight the importance of considering position-specific effects in codon optimization strategies.

Dependencies

Download R’s dependencies using RStudio’s built in functionality or your preferred method from The Comprehensive R Archive Network (https://cran.r-project.org/), Bioconductor (https://bioconductor.org/), and/or GitHub (https://github.com/).

The primary input for this pipeline is a codon multiple sequence alignment. We provide a detailed procedure to generate a human anchored codon multiple sequence alignment with Discontiguous Megablast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and Multiple Alignment of Coding Sequences Accounting for Frameshifts and Stop Codons (MACSE – GE²pop (agap-ge2pop.org)) in our manuscript, but the pipeline is compatible with any codon multiple sequence alignment.

Archived Datasets

Supplemental File 2, File 3, File 4, and File 5 (Supplementary Figures S1, S2, S3, S4): Human reference alignment’s visualization with NCBI’s Multiple sequence alignment viewer for ADAMTS13, F8, F9, and VWF, respectively. Sequence ID corresponds to Gene Accession Number and Start and End indicates the first and last nucleotide number of each sequence. Red lines indicate matching nucleotides, gray lines indicate unmatching nucleotides, and white lines indicate gaps.

• Supplemental File 2.svg is _ADAMTS13’_s plot.
• Supplemental File 3.svg is Factor 8’s plot.
• Supplemental File 4.svg is Factor 9’s plot.
• Supplemental File 5.svg is Von Willebrand Factor’s plot.

Following the Multiple Sequence Alignment Viewer user guide, the human reference sequences were set as anchor sequences, because they were used for the Discontiguous Megablast Searches (NCBI 2023). An anchor sequence subsets an alignment to compare the anchor sequence with other sequences.

Supplemental File 6.xlsx (Supplementary Table S2):

Alignment- and Position-specific codon-pair usages and codon-pair usage bias parameters including RSCPF, CPS, and CPOE, for four human reference genes. Codon-pair position number starts with “codons 1 and 2” as “codon-pair position 1”. PTM: post translational modification. Amino acid pair conservation was estimated as the average score of adjacent conservation of amino acids by ConSurf. The “Synonymous?” column indicates how many codon(s) in the pair are synonymous to human the human codon-pair (0, 1, 2). CPS’s amino Nacid/amino acid pair term (Fmn/Fm·Fn) is reported for both alignment-specific and position-specific analyses. The “Optimal codon-pair” column describes each position-specific codon-pair with respect to the most observed amino acid pair (defined as optimal). A codon-pair was “position-specific” optimal if it has the highest observed RSCPF at that position. A codon-pair was “alignment-specific” optimal if it has the highest observed RSCPF in the alignment.

Supplemental File 7.xlsx (Supplementary Table S3):

Sequence- and Position-specific codon usage tables for four human reference genes.

Archived Scripts

The scripts are listed by the order they should be ran. Some of the scripts are computationally intensive and will have the best performance on a high-performance computing (HPC) platform. Computationally intensive scripts have “HPC” in their names, and the rest of the scripts have “Local” in their names.

• Local – transcript selection per species.R

This script extracts a single transcript per species from Discontiguous Megablast results (fasta, hit-table, descriptions, and GenBank).

• Local - Codon-alignment-validation.R

This script compares the codon usage of sequences from a codon alignment to their original sequences.

• HPC - Permuted_alignment_generator.R

This script generates random alignments using the positional and sequential random codon sampling methods from an input codon alignment.

• HPC - Permutation codon-pair SU.R

This script calculates the codon pair symmetric uncertainty test statistic for each random and original alignment.

• Local - SU test statistic plots.R

This script calculates the codon pair symmetric uncertainty test statistic for the input alignment.

• Local - SU functions.R

Helper functions for Local - SU test statistic plots.R and Local – Codon-pair bias metric figures.R.

• Local – Alignment-specific CPOE.R

This script calculates alignment-specific codon-pair observed to expected ratios (CPOE) for an input alignment.

• Local – Alignment-specific RSCPF and CPS.R

This script calculates alignment-specific relative synonymous codon-pair frequency (RSCPF) and codon-pair score (CPS) for an input alignment.

• Local – Position-specific RSCPF-CPS-CPOE.R

This script calculates position-specific relative synonymous codon-pair frequency (RSCPF), codon-pair score (CPS), and codon-pair observed to expected ratio (CPOE) for an input alignment.

• Local – Codon-pair bias metric figures.R

This script creates the figures from the manuscript for an input alignment.

• Local – analysis of position-specific optimal codon-pairs.R

This script performs the naïve codon-pair optimization algorithm and analysis discussed in Table 5.

Authors

Nathan J. Clement ^1,+, Nobuko Hamasaki-Katagiri ^1,+, Brian Lin ^1,+, Anton A. Komar ², Michael DiCuccio ³, Haim Bar ⁴, and Chava Kimchi-Sarfaty ^{1, *}

Affiliations

¹ Hemostasis Branch 1, Division of Hemostasis, Office of Plasma Protein Therapeutics, Office Therapeutic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.

² Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA.

³ Rockville, MD 20853, USA.

⁴ Department of Statistics, University of Connecticut, Storrs, CT 06269, USA.

⁺ Equal contributor

^* Corresponding author

Please direct questions to Nathan Clement ([email protected]) and Chava Kimchi-Sarfaty ([email protected]).

Open-Source Disclaimer

This open-source software has been released as-is without expectation of support or future releases.