Abstract:Standard units of measurement are required for the quantitative description of nature; however, few standard units have been established for genomics to date. Here, we have developed a synthetic DNA ladder that defines a quantitative standard unit that can measure DNA sequence abundance within a next-generation sequencing library. The ladder can be spiked into a DNA sample, and act as an internal scale that measures quantitative genetics features. Unlike previous spike-ins, the ladder is encoded within a singl… Show more
“…This CAPTOR master mixture was then used to prepare libraries from mock microbial communities for ONT sequencing (as described above). The variable CAPTOR sequences were then retrieved from each read, counted and compared to the expected CAPTOR concentration to generate a staggered reference ladder that can measure quantitative library features 22 (see Methods). Fig.…”
Library adaptors are short oligonucleotides that are attached to RNA and DNA samples in preparation for next-generation sequencing (NGS). Adaptors can also include additional functional elements, such as sample indexes and unique molecular identifiers, to improve library analysis. Here, we describe Control Library Adaptors, termed CAPTORs, that measure the accuracy and reliability of NGS. CAPTORs can be integrated within the library preparation of RNA and DNA samples, and their encoded information is retrieved during sequencing. We show how CAPTORs can measure the accuracy of nanopore sequencing, evaluate the quantitative performance of metagenomic and RNA sequencing, and improve normalisation between samples. CAPTORs can also be customised for clinical diagnoses, correcting systematic sequencing errors and improving the diagnosis of pathogenic BRCA1/2 variants in breast cancer. CAPTORs are a simple and effective method to increase the accuracy and reliability of NGS, enabling comparisons between samples, reagents and laboratories, and supporting the use of nanopore sequencing for clinical diagnosis.
“…This CAPTOR master mixture was then used to prepare libraries from mock microbial communities for ONT sequencing (as described above). The variable CAPTOR sequences were then retrieved from each read, counted and compared to the expected CAPTOR concentration to generate a staggered reference ladder that can measure quantitative library features 22 (see Methods). Fig.…”
Library adaptors are short oligonucleotides that are attached to RNA and DNA samples in preparation for next-generation sequencing (NGS). Adaptors can also include additional functional elements, such as sample indexes and unique molecular identifiers, to improve library analysis. Here, we describe Control Library Adaptors, termed CAPTORs, that measure the accuracy and reliability of NGS. CAPTORs can be integrated within the library preparation of RNA and DNA samples, and their encoded information is retrieved during sequencing. We show how CAPTORs can measure the accuracy of nanopore sequencing, evaluate the quantitative performance of metagenomic and RNA sequencing, and improve normalisation between samples. CAPTORs can also be customised for clinical diagnoses, correcting systematic sequencing errors and improving the diagnosis of pathogenic BRCA1/2 variants in breast cancer. CAPTORs are a simple and effective method to increase the accuracy and reliability of NGS, enabling comparisons between samples, reagents and laboratories, and supporting the use of nanopore sequencing for clinical diagnosis.
“…Spike-in standards facilitate the quantification of microbial targets in metagenomes at accuracies similar to quantitative PCR methods (6)(7)(8)11,12). The major advantage of quantitative metagenomics is the ability to simultaneously quantify thousands of targets.…”
Section: Discussionmentioning
confidence: 99%
“…Both of these approaches depend on the accuracy of time-and resource-consuming ancillary measurements that can be error prone and the latter is not suitable for quantitative viral metagenomes (viromes), as viruses lack conserved genes. Alternatively, DNA standards have been added to samples at known concentrations to facilitate absolute abundance measurements in metagenomes (6)(7)(8)(9)(10)(11)(12)(13)(14)(15). With this approach, target gene or genome counts in the metagenome are quantified using the number of reads that map to the standards and the known standard concentrations (6,8,(11)(12)(13)15).…”
Section: Introductionmentioning
confidence: 99%
“…Alternatively, DNA standards have been added to samples at known concentrations to facilitate absolute abundance measurements in metagenomes (6)(7)(8)(9)(10)(11)(12)(13)(14)(15). With this approach, target gene or genome counts in the metagenome are quantified using the number of reads that map to the standards and the known standard concentrations (6,8,(11)(12)(13)15).…”
Section: Introductionmentioning
confidence: 99%
“…Of these, the synthetic standards have a number of benefits that contribute to their rising popularity. Synthetic DNA standards avoid non-specific mapping, as the nonsense sequences used are unique from known organisms (6,8), they can be designed to reflect the range of characteristics expected in a sampled microbiome, including input DNA lengths, GC contents, and concentrations, and they are readily available in premade mixtures (16). They have been used to assess the temporal and spatial variability of microbial populations in healthcare environments (9), antibiotic resistance genes and pathogens in wastewater (10) and manure (11), and microbial community structure and function in saltmarshes (7).…”
Quantitative metagenomic methods are maturing but continue to lack clearly-defined analytical limits. We used synthetic ssDNA and dsDNA standards to develop a quantitative metagenomic approach, called QuantMeta, that accounts for detection and quantification limitations. QuantMeta applies entropy-based detection limits that incorporate both read distribution and coverage and sets read depth variability thresholds to detect and correct quantification errors caused by non-specific mapping and assembly errors. In proof-of-concept wastewater metagenomes, we compared QuantMeta-based total virus concentrations, as well as polyomaviruses and crAss-like phages of interest, to those of traditional, low-throughput methods. The QuantMeta approach, applicable to both viral and cellular metagenomes, advances quantitative metagenomics by improving the accuracy of measured target concentrations.
High-throughput technologies for multiomics or molecular phenomics profiling have been extensively adopted in biomedical research and clinical applications, offering a more comprehensive understanding of biological processes and diseases. Omics reference materials play a pivotal role in ensuring the accuracy, reliability, and comparability of laboratory measurements and analyses. However, the current application of omics reference materials has revealed several issues, including inappropriate selection and underutilization, leading to inconsistencies across laboratories. This review aims to address these concerns by emphasizing the importance of well-characterized reference materials at each level of omics, encompassing (epi-)genomics, transcriptomics, proteomics, and metabolomics. By summarizing their characteristics, advantages, and limitations along with appropriate performance metrics pertinent to study purposes, we provide an overview of how omics reference materials can enhance data quality and data integration, thus fostering robust scientific investigations with omics technologies.
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