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Article ; Online: A universal molecular control for DNA, mRNA and protein expression.

Gunter, Helen M / Youlten, Scott E / Reis, Andre L M / McCubbin, Tim / Madala, Bindu Swapna / Wong, Ted / Stevanovski, Igor / Cipponi, Arcadi / Deveson, Ira W / Santini, Nadia S / Kummerfeld, Sarah / Croucher, Peter I / Marcellin, Esteban / Mercer, Tim R

Nature communications

2024 Volume 15, Issue 1, Page(s) 2480

Abstract: The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are ... ...

Abstract	The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.
MeSH term(s)	RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Proteomics ; DNA/genetics ; Genomics ; RNA
Chemical Substances	RNA, Messenger ; DNA (9007-49-2) ; RNA (63231-63-0)
Language	English
Publishing date	2024-03-20
Publishing country	England
Document type	Journal Article
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-024-46456-9
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests.

Madala, Bindu Swapna / Reis, Andre L M / Deveson, Ira W / Rawlinson, William / Mercer, Tim R

Scientific reports

2021 Volume 11, Issue 1, Page(s) 2636

Abstract: DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target ... ...

Abstract	DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. However, negative controls are also required to evaluate test specificity. Using a pair of chimeric A/B RNA standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The chimeric standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. This design enables cross-validation of positive and negative controls between the paired standards in the same reaction, with identical conditions. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. The chimeric A/B standards were assessed using the US Centres for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS-CoV-2 in patient samples. This chimeric reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms.
MeSH term(s)	Amino Acid Sequence ; COVID-19/diagnosis ; COVID-19/virology ; Chimera ; Humans ; RNA, Viral/standards ; Reference Standards ; Reproducibility of Results ; SARS-CoV-2/chemistry ; SARS-CoV-2/genetics ; SARS-CoV-2/isolation & purification ; Sensitivity and Specificity
Chemical Substances	RNA, Viral
Language	English
Publishing date	2021-01-29
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-021-81760-0
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing

Helen M. Gunter / Scott E. Youlten / Bindu Swapna Madala / Andre L. M. Reis / Igor Stevanovski / Ted Wong / Sarah K. Kummerfield / Ira W. Deveson / Nadia S. Santini / Esteban Marcellin / Tim R. Mercer

Nature Communications, Vol 13, Iss 1, Pp 1-

2022 Volume 11

Abstract: Adding library adaptors to DNA samples is an essential step in preparing samples for next-generation sequencing. Here, Gunter et al. describe the development of Control Library Adaptors (CAPTORs), that correct sequencing errors and normalise quantitative ...

Abstract	Adding library adaptors to DNA samples is an essential step in preparing samples for next-generation sequencing. Here, Gunter et al. describe the development of Control Library Adaptors (CAPTORs), that correct sequencing errors and normalise quantitative biases in Nanopore libraries.
Keywords	Science ; Q
Language	English
Publishing date	2022-10-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests

Bindu Swapna Madala / Andre L. M. Reis / Ira W. Deveson / William Rawlinson / Tim R. Mercer

Scientific Reports, Vol 11, Iss 1, Pp 1-

2021 Volume 8

Abstract: Abstract DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent ... ...

Abstract	Abstract DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. However, negative controls are also required to evaluate test specificity. Using a pair of chimeric A/B RNA standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The chimeric standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. This design enables cross-validation of positive and negative controls between the paired standards in the same reaction, with identical conditions. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. The chimeric A/B standards were assessed using the US Centres for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS-CoV-2 in patient samples. This chimeric reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms.
Keywords	Medicine ; R ; Science ; Q
Subject code	380
Language	English
Publishing date	2021-01-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Using synthetic chromosome controls to evaluate the sequencing of difficult regions within the human genome

Reis, Andre L. M. / Deveson, Ira W. / Madala, Bindu Swapna / Wong, Ted / Barker, Chris / Xu, Joshua / Lennon, Niall / Tong, Weida / Mercer, Tim R.

Genome biology. 2022 Dec., v. 23, no. 1

2022

Abstract: BACKGROUND: Next-generation sequencing (NGS) can identify mutations in the human genome that cause disease and has been widely adopted in clinical diagnosis. However, the human genome contains many polymorphic, low-complexity, and repetitive regions that ...

Institution	on behalf of the SEQC2 Consortium
Abstract	BACKGROUND: Next-generation sequencing (NGS) can identify mutations in the human genome that cause disease and has been widely adopted in clinical diagnosis. However, the human genome contains many polymorphic, low-complexity, and repetitive regions that are difficult to sequence and analyze. Despite their difficulty, these regions include many clinically important sequences that can inform the treatment of human diseases and improve the diagnostic yield of NGS. RESULTS: To evaluate the accuracy by which these difficult regions are analyzed with NGS, we built an in silico decoy chromosome, along with corresponding synthetic DNA reference controls, that encode difficult and clinically important human genome regions, including repeats, microsatellites, HLA genes, and immune receptors. These controls provide a known ground-truth reference against which to measure the performance of diverse sequencing technologies, reagents, and bioinformatic tools. Using this approach, we provide a comprehensive evaluation of short- and long-read sequencing instruments, library preparation methods, and software tools and identify the errors and systematic bias that confound our resolution of these remaining difficult regions. CONCLUSIONS: This study provides an analytical validation of diagnosis using NGS in difficult regions of the human genome and highlights the challenges that remain to resolve these difficult regions.
Keywords	DNA ; bioinformatics ; chromosomes ; computer simulation ; computer software ; humans ; microsatellite repeats
Language	English
Dates of publication	2022-12
Size	p. 19.
Publishing place	BioMed Central
Document type	Article
ZDB-ID	2040529-7
ISSN	1474-760X
ISSN	1474-760X
DOI	10.1186/s13059-021-02579-6
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Using synthetic chromosome controls to evaluate the sequencing of difficult regions within the human genome.

Reis, Andre L M / Deveson, Ira W / Madala, Bindu Swapna / Wong, Ted / Barker, Chris / Xu, Joshua / Lennon, Niall / Tong, Weida / Mercer, Tim R

Genome biology

2022 Volume 23, Issue 1, Page(s) 19

Abstract: Background: Next-generation sequencing (NGS) can identify mutations in the human genome that cause disease and has been widely adopted in clinical diagnosis. However, the human genome contains many polymorphic, low-complexity, and repetitive regions ... ...

Abstract	Background: Next-generation sequencing (NGS) can identify mutations in the human genome that cause disease and has been widely adopted in clinical diagnosis. However, the human genome contains many polymorphic, low-complexity, and repetitive regions that are difficult to sequence and analyze. Despite their difficulty, these regions include many clinically important sequences that can inform the treatment of human diseases and improve the diagnostic yield of NGS. Results: To evaluate the accuracy by which these difficult regions are analyzed with NGS, we built an in silico decoy chromosome, along with corresponding synthetic DNA reference controls, that encode difficult and clinically important human genome regions, including repeats, microsatellites, HLA genes, and immune receptors. These controls provide a known ground-truth reference against which to measure the performance of diverse sequencing technologies, reagents, and bioinformatic tools. Using this approach, we provide a comprehensive evaluation of short- and long-read sequencing instruments, library preparation methods, and software tools and identify the errors and systematic bias that confound our resolution of these remaining difficult regions. Conclusions: This study provides an analytical validation of diagnosis using NGS in difficult regions of the human genome and highlights the challenges that remain to resolve these difficult regions.
MeSH term(s)	Chromosomes ; Genome, Human ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Microsatellite Repeats ; Sequence Analysis, DNA/methods ; Software
Language	English
Publishing date	2022-01-12
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2040529-7
ISSN	1474-760X ; 1474-760X
ISSN (online)	1474-760X
ISSN	1474-760X
DOI	10.1186/s13059-021-02579-6
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing.

Gunter, Helen M / Youlten, Scott E / Madala, Bindu Swapna / Reis, Andre L M / Stevanovski, Igor / Wong, Ted / Kummerfield, Sarah K / Deveson, Ira W / Santini, Nadia S / Marcellin, Esteban / Mercer, Tim R

Nature communications

2022 Volume 13, Issue 1, Page(s) 6437

Abstract: 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, ... ...

Abstract	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.
MeSH term(s)	Nanopore Sequencing ; Reproducibility of Results ; Gene Library ; High-Throughput Nucleotide Sequencing/methods ; RNA
Chemical Substances	RNA (63231-63-0)
Language	English
Publishing date	2022-10-28
Publishing country	England
Document type	Journal Article
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-022-34028-8
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: A universal and independent synthetic DNA ladder for the quantitative measurement of genomic features.

Reis, Andre L M / Deveson, Ira W / Wong, Ted / Madala, Bindu Swapna / Barker, Chris / Blackburn, James / Marcellin, Esteban / Mercer, Tim R

Nature communications

2020 Volume 11, Issue 1, Page(s) 3609

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 ... ...

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 single molecule, and can be equivalently and independently synthesized by different laboratories. We show how the ladder can measure diverse quantitative features, including human genetic variation and microbial abundance, and also estimate uncertainty due to technical variation and improve normalization between libraries. This ladder provides an independent quantitative unit that can be used with any organism, application or technology, thereby providing a common metric by which genomes can be measured.
MeSH term(s)	Base Sequence ; DNA/analysis ; DNA/chemical synthesis ; DNA/genetics ; Gene Dosage ; Gene Library ; Genomics ; Humans
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2020-07-17
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-020-17445-5
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Using synthetic chromosome controls to evaluate the sequencing of difficult regions within the human genome

Andre L. M. Reis / Ira W. Deveson / Bindu Swapna Madala / Ted Wong / Chris Barker / Joshua Xu / Niall Lennon / Weida Tong / Tim R. Mercer / on behalf of the SEQC2 Consortium

Genome Biology, Vol 23, Iss 1, Pp 1-

2022 Volume 24

Abstract: Abstract Background Next-generation sequencing (NGS) can identify mutations in the human genome that cause disease and has been widely adopted in clinical diagnosis. However, the human genome contains many polymorphic, low-complexity, and repetitive ... ...

Abstract	Abstract Background Next-generation sequencing (NGS) can identify mutations in the human genome that cause disease and has been widely adopted in clinical diagnosis. However, the human genome contains many polymorphic, low-complexity, and repetitive regions that are difficult to sequence and analyze. Despite their difficulty, these regions include many clinically important sequences that can inform the treatment of human diseases and improve the diagnostic yield of NGS. Results To evaluate the accuracy by which these difficult regions are analyzed with NGS, we built an in silico decoy chromosome, along with corresponding synthetic DNA reference controls, that encode difficult and clinically important human genome regions, including repeats, microsatellites, HLA genes, and immune receptors. These controls provide a known ground-truth reference against which to measure the performance of diverse sequencing technologies, reagents, and bioinformatic tools. Using this approach, we provide a comprehensive evaluation of short- and long-read sequencing instruments, library preparation methods, and software tools and identify the errors and systematic bias that confound our resolution of these remaining difficult regions. Conclusions This study provides an analytical validation of diagnosis using NGS in difficult regions of the human genome and highlights the challenges that remain to resolve these difficult regions.
Keywords	Biology (General) ; QH301-705.5 ; Genetics ; QH426-470
Language	English
Publishing date	2022-01-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests

Madala, Bindu Swapna / Reis, Andre L. M. / Deveson, Ira W. / Rawlinson, William / Mercer, Tim R.

bioRxiv

Abstract	DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. However, negative controls are also required to evaluate test specificity. Using a pair of chimeric A/B RNA standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The chimeric standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. This design enables cross-validation of positive and negative controls between the paired standards in the same reaction, with identical conditions. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. The chimeric A/B standards were assessed using the US Centers for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS CoV-2 in patient samples. This chimeric reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms.
Keywords	covid19
Publisher	BioRxiv; WHO
Document type	Article ; Online
DOI	10.1101/2020.06.09.143412
Database	COVID19

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