Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing

Abstract

Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.

Gene Variant of Barrier to Autointegration Factor 2 (Banf2w) Is Concordant with Female Determination in Cichlids

“…However, unlike the female sequence assembly (SRA accession number: ERX2240357), in this case (SRA accession number: SRX7899544), only three haplotypes were found. Moreover, sequence chromatograms of some of the specimens pointed to additional types of females with presumably three banf2 copies, in accordance with the decomposition analysis performed by Tide software (Figures 3b and 4b and Figure S3) [52,53]. We were also able to distinguish male types in Sanger sequencing by an informative SNP within exon 3, which allowed deduction of the gene copy proportion by the peak signal ratio of the C/T nucleotides (Figure 4).…”

“…Indeed, peak-height analyses of Sanger chromatograms of some female individuals from the hybrid Oa stock also supported this model (Figure 4a). For these specimens, banf2w represented approximately 25% of the trace signal according to Tide software [52,53], suggesting a 3:1 ratio for banf2/ban2fw (Figure S3). According to this model, banf2w and banf2c are located on the W chromosome, while banf2a and banf2b are situated on the Z chromosome.…”

“…Copy numbers were estimated taking into account coverage of next generation sequencing (NGS) reads, and copy number proportion was measured by Sanger chromatograms [52]. Proportions of copies were estimated using Tide software [52,53].…”

“…Copy numbers were estimated taking into account coverage of next generation sequencing (NGS) reads, and copy number proportion was measured by Sanger chromatograms [52]. Proportions of copies were estimated using Tide software [52,53]. Each chromatogram analysis was performed using template DNA of at least six individuals with the same sex phenotype and genotypes.…”

Cross‐species conservation of a transposase‐linked element enables genetic sexing of commercial populations of Russian sturgeon (Acipenser gueldenstaedtii)

“…This amplification product, when extracted from agarose gel and sequenced showed that this gene has multiple copies (Figure 2b,c). Investigation of copy-number variation based on Tide analysis for individual fish that had a shift in their sequence (Brinkman et al, 2014;Seroussi, 2021), and peak ratio examination of four SNPs for other individual fish (Fig. S1, Seroussi, 2021) suggested that this gene is in keeping with an octaploid model (Figure 2b,c).…”