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Calculating peak-height ratios between SNP alleles in sequencing chromatograms is a practical method for estimating their copy-number proportions. However, it is surprising that sequencing DNA from different directions might yield different results. We analyzed three adjacent SNPs within the ovine period circadian-clock 2 (PER2) gene that displayed...
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... For all tested amplicons in this study, the accuracy of Sanger sequencing was further enhanced by genotyping additional variation that happened to occur within the same chromatogram near the target SNPs. This validation also allowed estimating copy-number proportions based on the chromatogram's double peak ratios [23] and ruling out copy-number variation, concluding that uneven peaks ( Figure S2) were likely the results of a secondary structure that may have promoted wobble-like pairing [24]. ...
In dairy cattle, identifying polymorphisms that contribute to complex economical traits such as residual feed intake (RFI) is challenging and demands accurate genotyping. In this study, we compared imputed genotypes (n = 192 cows) to those obtained using the TaqMan and high-resolution melting (HRM) methods (n = 114 cows), for mutations in the FABP4 gene that had been suggested to have a large effect on RFI. Combining the whole genome sequence (n = 19 bulls) and the cows’ BovineHD BeadChip allowed imputing genotypes for these mutations that were verified by Sanger sequencing, whereas, an error rate of 11.6% and 10.7% were encountered for HRM and TaqMan, respectively. We show that this error rate seriously affected the linkage-disequilibrium analysis that supported this gene candidacy over other BTA14 gene candidates. Thus, imputation produced superior genotypes and should also be regarded as a method of choice to validate the reliability of the genotypes obtained by other methodologies that are prone to genotyping errors due to technical conditions. These results support the view that RFI is a complex trait and that searching for the causative sequence variation underlying cattle RFI should await the development of statistical methods suitable to handle additive and epistatic interactions.
... Termed replication diode, complete absence has been shown to arise from the presence of stem-loop structures capable of guanine (G)-T wobblepairing within the tested amplicon. Stabilization of these structures for specific alleles in heterozygous situations mediates the orientation bias by hindering DNA polymerase passage on one strand, while, on the complementary strand, the non-paired adenine (A)-C nucleotide counterparts allow unobstructed replication [13]. Thus, bidirectional sequencing is mandatory for heterozygote detection. ...
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.
... Calculating peak-height ratios between SNP alleles in sequencing chromatograms is another practical method for estimating their copy number proportions, especially when similar ratios are obtained by sequencing from the forward and reverse orientations [25,26]. To better characterize v7, which was common in Holsteins (75% of individuals, Supplementary Table S3), we compared the sequence assembly of FCGR2 Ex3 based on next-generation sequencing to the Sanger-sequencing chromatograms of this exon from an influential Israeli AI sire. ...
Differentiation of cells by flow cytometry provides informative somatic cell counts (SCCs) that allow analyzing leukocyte population patterns in udder infections of different etiologies. Postulating that this approach also enhances the statistical power to detect genetic variants linked to cell levels in milk of healthy mammary glands, we used monoclonal antibodies anti-CD18, anti-CD4, anti--CD14, and anti-PMN to count cells presenting these surface antigens, and performed a genome-wide association study of these counts in 125 Israeli Holsteins genotyped using SNP BeadChips. We identified an informative haplotype of 15 SNPs in the centromeric end of BTA3 that was strongly associated with CD18 cells (p < 2.3 × 10−9). Within this region, examination of the network of genes interacting with ITGB2 (CD18) indicated an Fc-γ-receptor gene cluster, including FCGR2A (CD32). Sanger-sequence analysis of FCGR2s-linked exon 3 variation to CD18 counts. Meta-analysis of RNA-Seq data revealed a significant negative correlation (R = −0.51) between expression of CD32 and CD18 in milk. Assembly of DNA-Seq reads uncovered FCGR copy-number variation and a variant, designated V7, was abundant in dairy cattle, probably reflecting adaptation to selection pressure for low SCC in Holstein milk.
... 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 In this study, we demonstrate the original innovation only by showing encoding words, but our platform can be scaled up by using automated droplet microfluidics in parallel for writing and parallel nanopore measurement for reading. 35 The amplification of these DNA nanostructures is possible if we optimize the design by using closed ssDNA bulges or DNA stem loops 36,37 instead of the open overhangs. Our DNA-HDs hence offer the potential to copy data including random data access by target amplification. ...
Nanopores are powerful single-molecule tools for label-free sensing of nanoscale molecules including DNA, which can be used for building designed nanostructures and performing computations. Here, DNA hard drives (DNA-HDs) are introduced based on DNA nanotechnology and nanopore sensing as a rewritable molecular memory system, allowing for storing, operating and reading data in the changeable three-dimensional structure of DNA. Writing and erasing data are significantly improved compared to previous molecular storage systems by employing controllable attachment and removal of molecules on a long double-stranded DNA. Data reading is achieved by detecting the single molecules at the microsecond timescale using nanopores. The DNA-HD also ensures secure data storage where the data can only be read after providing the correct physical molecular keys. Our approach allows for easy-writing and easy-reading, rewritable and secure data storage toward a promising miniature scale integration for molecular data storage and computation.
... Detection and characterization of indels was performed using ShiftDetector and the ABI tracefiles (Seroussi et al. 2002). Copy number proportions were estimated based on the ABI chromatograms peak-height ratios (Seroussi et al. 2013;Shirak et al. 2017). ...
Effective farming of tilapia requires all-male culture, characterized by uniformity and high growth rate. Males of O. aureus (Oa) and females of O. niloticus (On) produce all-male offspring, but there is a behavioral reproductive barrier between the two species that prevents mass production. In crosses between Oa and On broodstocks, few hybrid females are attracted to the Oa male nests (denoted responders), and if they harbor the On alleles for the sex determination (SD) sites on linkage groups (LGs) 1, 3, and 23, all-male progeny are produced. Yet, without controlling for the alleles underlying SD, the parental stocks gradually lose their capability for all-male production. Hypothesizing that marker-assisted selection for female responders would allow production of sustainable broodstocks, we applied genotyping-by-sequencing to generate 4983 informative SNPs from 13 responding and 28 non-responding females from two full-sib families. Accounting for multiple comparisons in a genome-wide association study, seven SNPs met a false discovery rate of 0.061. Lowest nominal probabilities were on LGs 9 and 14, for which microsatellite DNA markers were designed within the candidate genes PTGDSL and CASRL, respectively. By increasing the sample size to 22 responders and 47 non-responders and by genotyping additional established microsatellites, we confirmed the association of these LGs with female responsiveness. The combined effects of microsatellites GM171 and CARSL-LOC100690618 on LGs 9 and 14 explained 37% of the phenotypic variance of reproductive interaction (p < 0.0001). Based on these findings, we propose a strategy for mass production of all-male tilapia hybrids through selection for genomic loci affecting SD and female responsiveness.
