AdvISER-PYRO: Amplicon Identification using SparsE Representation of PYROsequencing signal

Ambroise, Jérôme; Piette, Anne-Sophie; Delcorps, Cathy; Rigouts, Leen; Jong, Bouke C. de; Irenge, Léonid M.; Robert, Annie; Gala, Jean‐Luc

doi:10.1093/bioinformatics/btt339

Cited by 8 publications

(10 citation statements)

References 17 publications

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“…The TEM mix contained 6, 20 and 60 pmol of the TEM-seq104, TEM-seq164 and TEM-seq238 primers, respectively, whilst the SHV mix contained 60 and 20 pmol of the SHV-seq179 and SHV-seq238 primers, respectively. The pyrosequencing nucleotide dispensation orders were selected using SENATOR (SElecting the Nucleotide dispensATion Order) algorithm (Ambroise et al 2013 ). Nucleotide dispensation orders with 15 (CAGCCTGACATATCA) and 11 (GTGACTGCGTC) nucleotides were selected for the triplex TEM and duplex SHV assays, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…All multiplex pyrosequencing signals generated for TEM and SHV were analyzed using a new web interactive application (available at https://ucl-irec-ctma.shinyapps.io/Pyrosequencing-TEM-SHV and in ‘download’ section of http://www.uclouvain.be/ctma.html where a template data set can be downloaded) written with R.3.1.2 software (Ihaka and Gentleman 1996 ) using the “shiny” package. This new application was developed using an improved version of three previously published algorithms including (1) AdvISER-PYRO for analyzing low and complex signals resulting from samples including several mycobacteria (Ambroise et al 2013 ), (2) AdvISER-M-PYRO for analyzing overlapping pyro-signals generated from multiplex reactions conducted on mono-allelic genes in bacteria (Ambroise et al 2014 ), and (3) AdvISER-MH-PYRO for analyzing overlapping pyro-signals generated from multiplex reactions to genotype bi-allelic human SNP (Ambroise et al 2015 ). While the initial version of AdvISER-M-PYRO analyzed multiplex pyrosequencing signals by selecting a single and unique sequence for each genomic region, this new version enables the analysis of multiplex pyrosequencing signals generated from samples including two distinct sequences (i.e., plasmid and chromosomal) for each genomic region.…”

Section: Methodsmentioning

confidence: 99%

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A qPCR and multiplex pyrosequencing assay combined with automated data processing for rapid and unambiguous detection of ESBL-producers Enterobacteriaceae

Deccache¹,

Irenge²,

Ambroise

et al. 2015

AMB Expr

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Rapid and specific detection of extended-spectrum β-lactamase-producing (ESBL) bacteria is crucial both for timely antibiotic therapy when treating infected patients as well as for appropriate infection control measures aimed at curbing the spread of ESBL-producing isolates. Whereas a variety of phenotypic methods are currently available for ESBL detection, they remain time consuming and sometimes difficult to interpret while being also affected by a lack of sensitivity and specificity. Considering the longer turnaround time (TAT) of susceptibility testing and culture results, DNA-based ESBL identification would be a valuable surrogate for phenotypic-based methods. Putative ESBL-positive Enterobacteriaceae isolates (n = 330) from clinical specimen were prospectively collected in Bulgaria, Romania and Democratic Republic of Congo and tested in this study. All isolates were assessed for ESBL-production by the E-test method and those giving undetermined ESBL status were re-tested using the combination disk test. A genotypic assay successively combining qPCR detection of blaCTX-M, blaTEM and blaSHV genes with a multiplex pyrosequencing of blaTEM and blaSHV genes was developed in order to detect the most common ESBL-associated TEM and SHV single nucleotides polymorphisms, irrespective of their plasmid and/or chromosomal location. This assay was applied on all Enterobacteriaceae isolates (n = 330). Phenotypic and genotypic results matched in 324/330 (98.2%). Accordingly, real-time PCR combined with multiplex pyrosequencing appears to be a reliable and easy-to-perform assay with high-throughput identification and fast TAT (~5 h).

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A qPCR and multiplex pyrosequencing assay combined with automated data processing for rapid and unambiguous detection of ESBL-producers Enterobacteriaceae

Deccache¹,

Irenge²,

Ambroise

et al. 2015

AMB Expr

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“…This technique can convert SAS into correct single sequence and translate MAS signals into the correct sequence pair compared to the pyrosequencing software. The software can be implemented in an R package and used in broad range of clinical applications in heterogenous tumour cell samples [ 66 ]. This traditional sequencing method is not considered to be powerful enough for standard sequencing needs because of the short read-lengths it generates to detect SNPs.…”

Section: Low-throughput Methodsmentioning

confidence: 99%

Single Nucleotide Polymorphisms (SNPs)

Batra

Srinivasan

Clements

2014

Molecular Testing in Cancer

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“…These methods were applied, inter alia, on gene expression data to classify tumors [ 7 ], on miRNA and mRNA expression data for glioblastoma subtyping [ 8 ], and on single nucleotide polymorphisms (SNP) and functional magnetic resonance imaging (fMRI) voxels to discriminate between schizophrenia cases and controls [ 6 ]. Regarding pyrosequencing analysis, sparse representation via constraint-based regression method was recently used to develop three complementary software solutions: (i) the AdvISER-PYRO software for analyzing low and complex signals resulting from samples including several mycobacteria [ 9 ], (ii) the AdvISER-M-PYRO software for analyzing overlapping pyro-signals generated from multiplex reactions conducted on mono-allelic genes in bacteria [ 10 ], and (iii) the AdvISER-MH-PYRO software for analyzing overlapping pyro-signals generated from multiplex reactions to genotype bi-allelic human SNP [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

Using a constraint-based regression method for relative quantification of somatic mutations in pyrosequencing signals: a case for NRAS analysis

Ambroise

Badir

Nienhaus

et al. 2016

Algorithms Mol Biol

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BackgroundPyrosequencing Allele Quantification (AQ) is a cost-effective DNA sequencing method that can be used for detecting somatic mutations in formalin-fixed paraffin-embedded (FFPE) samples. The method displays a low turnaround time and a high sensitivity. Pyrosequencing suffers however from two main drawbacks including (i) low specificity and (ii) difficult signal interpretation when multiple mutations are reported in a hotspot genomic region.ResultsUsing a constraint-based regression method, the new AdvISER-PYRO-SMQ algorithm was developed in the current study and implemented into an R package. As a proof-of-concept, AdvISER-PYRO-SMQ was used to identify a set of 9 distinct point mutations affecting codon 61 of the NRAS oncogene. In parallel, a pyrosequencing assay using the Qiagen software and its AQ module was used to assess selectively the presence of a single point mutation (NRAS - Q61R-1) among the set of codon 61 mutations, and to analyze related pyrosequencing signals. AdvISER-PYRO-SMQ produced a lower limit of blank (0 %) than the AQ module of Qiagen software (5.1 %) and similar limit of detection were obtained for both software (5.6 vs 4.8 %). AdvISER-PYRO-SMQ was able to screen for the presence of 9 distinct mutations with a single pyrosequencing reaction whereas the AQ module was limited to screen a single mutation per reaction.ConclusionUsing a constraint-based regression method enables to analyze pyrosequencing signal and to detect multiple mutations within a hotspot genomic region with an optimal compromise between sensitivity and specificity. The AdvISER-PYRO-SMQ R package provides a generic tool which can be applied on a wide range of somatic mutations. Its implementation in a Shiny web interactive application (available at https://ucl-irec-ctma.shinyapps.io/Pyrosequencing-NRAS-61/) enables its use in research or clinical routine applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13015-016-0086-4) contains supplementary material, which is available to authorized users.

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AdvISER-PYRO: Amplicon Identification using SparsE Representation of PYROsequencing signal

Cited by 8 publications

References 17 publications

A qPCR and multiplex pyrosequencing assay combined with automated data processing for rapid and unambiguous detection of ESBL-producers Enterobacteriaceae

A qPCR and multiplex pyrosequencing assay combined with automated data processing for rapid and unambiguous detection of ESBL-producers Enterobacteriaceae

Single Nucleotide Polymorphisms (SNPs)

Using a constraint-based regression method for relative quantification of somatic mutations in pyrosequencing signals: a case for NRAS analysis

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