Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples

<p>Abstract</p> <p>Background</p> <p>Pathogens such as fungi, bacteria and especially viruses, are highly variable even within an individual host, intensifying the difficulty of distinguishing and accurately quantifying numerous allelic variants co-existing in a single nucleic acid sample. The majority of currently available techniques are based on real-time PCR or primer extension and often require multiplexing adjustments that impose a practical limitation of the number of alleles that can be monitored simultaneously at a single locus.</p> <p>Results</p> <p>Here, we describe a novel method that allows the simultaneous quantification of numerous allelic variants in a single reaction tube and without multiplexing. Quantitative Single-letter Sequencing (QSS) begins with a single PCR amplification step using a pair of primers flanking the polymorphic region of interest. Next, PCR products are submitted to single-letter sequencing with a fluorescently-labelled primer located upstream of the polymorphic region. The resulting monochromatic electropherogram shows numerous specific diagnostic peaks, attributable to specific variants, signifying their presence/absence in the DNA sample. Moreover, peak fluorescence can be quantified and used to estimate the frequency of the corresponding variant in the DNA population.</p> <p>Using engineered allelic markers in the genome of <it>Cauliflower mosaic virus</it>, we reliably monitored six different viral genotypes in DNA extracted from infected plants. Evaluation of the intrinsic variance of this method, as applied to both artificial plasmid DNA mixes and viral genome populations, demonstrates that QSS is a robust and reliable method of detection and quantification for variants with a relative frequency of between 0.05 and 1.</p> <p>Conclusion</p> <p>This simple method is easily transferable to many other biological systems and questions, including those involving high throughput analysis, and can be performed in any laboratory since it does not require specialized equipment.</p

Immuno-purification of a dimeric subcomplex of the respiratory NAHD-CoQ reductase of Rhodobacter capsulatus equivalent to the FP fraction of the mitochondrial complex I

AbstractThe Rhodobacter capsulatus genes encoding the NUOE and NUOF subunits, equivalent to the 24 kDa and 51 kDa subunits of the mammalian mitochondrial complex I, have been sequenced. According to the nucleotide sequence, the NUOE subunit is 389 amino acids long and has a molecular mass of 41.3 kDa. In comparison to the mitochondrial equivalent subunit, NUOE is extended at the C terminus by more than 150 amino acids. The NUOF subunit is 431 amino acids long and has a molecular mass of 47.1 kDa. A subcomplex containing both the NUOE and NUOF subunits was extracted by detergent treatment of R. capsulatus membranes and immuno-purified. This subcomplex is homologous to the mitochondrial FP fragment. Mass spectrometry after trypsin treatment of the NUOE subunit validates the atypical primary structure deduced from the sequence of the gene

Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples-3

T1-6 plasmids upstream of the polymorphic region. Adenine residues marked in green are theoretically expected to yield discriminating peaks; ther A residues are indicated in black. The expected position (in bp) of each base from the fluorescent sequencing primer is indicated at the top. (B) Experimentally observed positions of discriminating A-peaks (named VIT1.1 to VIT6.2 at the top) on sequence traces (not shown) from individually sequenced markers. Sequences of all six markers are slightly distorted in order to match the actual position of A peaks observed on individual electropherograms. The red A residues were each confirmed to yield a discriminating peak. Though not theoretically predicted to do so, the red-boxed As proved to yield discriminating peaks experimentally. The remaining green As failed to produce discriminating-peaks. Sequencing reactions of pCa-VIT1-6 were repeated three times, and the observed position of all peaks was highly reproducible: +/- 0.2 bp among repeats. At the bottom, the number in blue is the average position of the last A common to all sequences upstream of the differential markers. The numbers in red are the average positions of the observed discriminating peaks. (C) Single-letter Sequencing electropherogram from a mixed DNA solution containing all 6 markers. A mix of all 6 pCaVIT1-6 plasmids in equal amounts was used as a template for PCR and subsequent single-letter sequencing. All discriminating-peaks defined in B are indicated by arrows on the electropherogram. The shaded blue peak corresponds to the last common A. The scale at the top indicates the nucleotide position relative to that of the sequencing primer. The scale on the left is used for scoring the height of the peaks in arbitrary units provided by the STRand program. The two purple peaks correspond to molecular weight markers. # These peaks are artefacts. ‡ Though appearing as discriminating, these peaks actually overlap small artefactual peaks observed on at least one electropherogram from individually sequenced markers (not shown). They are thus not used further.<p><b>Copyright information:</b></p><p>Taken from "Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples"</p><p>http://www.biomedcentral.com/1471-2164/9/85</p><p>BMC Genomics 2008;9():85-85.</p><p>Published online 21 Feb 2008</p><p>PMCID:PMC2276495.</p><p></p

Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples-0

T1-6 plasmids upstream of the polymorphic region. Adenine residues marked in green are theoretically expected to yield discriminating peaks; ther A residues are indicated in black. The expected position (in bp) of each base from the fluorescent sequencing primer is indicated at the top. (B) Experimentally observed positions of discriminating A-peaks (named VIT1.1 to VIT6.2 at the top) on sequence traces (not shown) from individually sequenced markers. Sequences of all six markers are slightly distorted in order to match the actual position of A peaks observed on individual electropherograms. The red A residues were each confirmed to yield a discriminating peak. Though not theoretically predicted to do so, the red-boxed As proved to yield discriminating peaks experimentally. The remaining green As failed to produce discriminating-peaks. Sequencing reactions of pCa-VIT1-6 were repeated three times, and the observed position of all peaks was highly reproducible: +/- 0.2 bp among repeats. At the bottom, the number in blue is the average position of the last A common to all sequences upstream of the differential markers. The numbers in red are the average positions of the observed discriminating peaks. (C) Single-letter Sequencing electropherogram from a mixed DNA solution containing all 6 markers. A mix of all 6 pCaVIT1-6 plasmids in equal amounts was used as a template for PCR and subsequent single-letter sequencing. All discriminating-peaks defined in B are indicated by arrows on the electropherogram. The shaded blue peak corresponds to the last common A. The scale at the top indicates the nucleotide position relative to that of the sequencing primer. The scale on the left is used for scoring the height of the peaks in arbitrary units provided by the STRand program. The two purple peaks correspond to molecular weight markers. # These peaks are artefacts. ‡ Though appearing as discriminating, these peaks actually overlap small artefactual peaks observed on at least one electropherogram from individually sequenced markers (not shown). They are thus not used further.<p><b>Copyright information:</b></p><p>Taken from "Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples"</p><p>http://www.biomedcentral.com/1471-2164/9/85</p><p>BMC Genomics 2008;9():85-85.</p><p>Published online 21 Feb 2008</p><p>PMCID:PMC2276495.</p><p></p

Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples-1

, as they illustrate the worst and the best fit, respectively, between the recorded data and the polynomial regression function calculated using Excel (correlation coefficients R, are shown). Vertical bars represent standard deviation among repeats.<p><b>Copyright information:</b></p><p>Taken from "Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples"</p><p>http://www.biomedcentral.com/1471-2164/9/85</p><p>BMC Genomics 2008;9():85-85.</p><p>Published online 21 Feb 2008</p><p>PMCID:PMC2276495.</p><p></p

Towards reliable, informative, cost-effective genotyping tools for the toolbox of grain legume breeders

International audienceSingle nucleotide polymorphisms (SNPs) are the most frequent source of genetic variation in eukaryotic species. Having access to well-distributed SNPs across the genome, segregating between and within the diversity groups of each species, is key to genetic studies and applications. Recently, we took advantage of the advances in second-generation sequencing technologies and the development of bioinformatics tools to characterise genetic diversity panels in pea and faba bean, two grain legume species with large genomes (4.45Gb and 13Gb, respectively). We chose a target capture technology focusing on coding regions only because whole-genome resequencing is relatively expensive for species with large genomes and because polymorphisms capture in repetitive non-coding regions is difficult to achieve or to interpret. We generated exome-enriched genomic libraries for 240 and 248 pea and faba bean accessions, respectively. After paired-end Illumina sequencing, the reads were mapped to the latest pea and faba bean genome assemblies, allowing the discovery of approximately 2.2 and 1.75 million robust SNPs in the two panels, respectively. The development of these SNP resources paves the way for a wide range of applications requiring low, medium or high marker density. These data can be used directly to set up genome-wide association studies using the sequenced panels in both species, as has already been demonstrated for aphid resistance in pea. They can also be used for the exploitation of genetic resources and formarker- and genomic-assisted selection. Here, we show how the pea and faba bean research and breeding communities can benefit from recent developments, and we highlight the importance of common tailored genotyping tools that can be developed based on the available SNP sets. We also discuss the importance of identifying and incorporating functional markers related to traits of interest including biotic and abiotic stress resistance and seed and protein quality

Large Functional Range of Steady-State Levels of Nuclear and Mitochondrial Transcripts Coding for the Subunits of the Human Mitochondrial OXPHOS System

We have measured, by reverse transcription and real-time quantitative PCR, the steady-state levels of the mitochondrial and nuclear transcripts encoding several subunits of the human oxidative phosphorylation (OXPHOS) system, in different normal tissues (muscle, liver, trachea, and kidney) and in cultured cells (normal fibroblasts, 143B osteosarcoma cells, 143B206 ρ(0) cells). Five mitochondrial transcripts and nine nuclear transcripts were assessed. The measured amounts of these OXPHOS transcripts in muscle samples corroborated data obtained by others using the serial analysis of gene expression (SAGE) method to appraise gene expression in the same type of tissue. Steady-state levels for all the transcripts were found to range over more than two orders of magnitude. Most of the time, the mitochondrial H-strand transcripts were present at higher levels than the nuclear transcripts. The mitochondrial L-strand transcript ND6 was usually present at a low level. Cultured 143B cells contained significantly reduced amounts of mitochondrial transcripts in comparison with the tissue samples. In 143B206 ρ(0) cells, fully depleted of mitochondrial DNA, the levels of nuclear OXPHOS transcripts were not modified in comparison with the parental cells. This observation indicated that nuclear transcription is not coordinated with mitochondrial transcription. We also observed that in the different tissues and cells, there is a transcriptional coregulation of all the investigated nuclear genes. Nuclear OXPHOS gene expression seems to be finely regulated. [The following individual kindly provided reagents, samples, or unpublished information as indicated in the paper: G. Attardi.

Quantification of Mitochondrial DNA Deletion, Depletion, and Overreplication: Application to Diagnosis

Background: Many mitochondrial pathologies are quantitative disorders related to tissue-specific deletion, depletion, or overreplication of mitochondrial DNA (mtDNA). We developed an assay for the determination of mtDNA copy number by real-time quantitative PCR for the molecular diagnosis of such alterations. Methods: To determine altered mtDNA copy number in muscle from nine patients with single or multiple mtDNA deletions, we generated calibration curves from serial dilutions of cloned mtDNA probes specific to four different mitochondrial genes encoding either ribosomal(16S) or messenger (ND2, ND5, and ATPase6) RNAs, localized in different regions of the mtDNA sequence. This method was compared with quantification of radioactive signals from Southern-blot analysis.We also determined the mitochondrial-to-nuclear DNA ratio in muscle, liver, and cultured fibroblasts from a patient with mtDNA depletion and in liver from two patients with mtDNA overreplication.Results: Both methods quantified 5–76% of deleted mtDNA in muscle, 59–97% of mtDNA depletion in the tissues, and 1.7- to 4.1-fold mtDNA overreplication in liver. The data obtained were concordant, with a linear correlation coefficient (r2) between the two methods of 0.94, and indicated that quantitative PCR has a higher sensitivity than Southern-blot analysis. Conclusions: Real-time quantitative PCR can determine the copy number of either deleted or full-length mtDNA in patients with mitochondrial diseases and has advantages over classic Southern-blot analysis