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Transplant Virus Detection Using Multiplex Targeted Sequencing.

ABSTRACT: Background:Viral infections are a major cause of complications and death in solid organ and hematopoietic cell transplantation. Methods:We developed a multiplex viral sequencing assay (mVseq) to simultaneously detect 20 transplant-relevant DNA viruses from small clinical samples. The assay uses a single-tube multiplex PCR to amplify highly conserved virus genomic regions without the need for previous virus enrichment or host nucleic acid subtraction. Multiplex sample sequencing was performed using Illumina MiSeq, and reads were aligned to a database of target sequences. Analytical and clinical performance was evaluated using reference viruses spiked into human plasma, as well as patient plasma and nonplasma samples, including bronchoalveolar lavage fluid, cerebrospinal fluid, urine, and tissue from immunocompromised transplant recipients. Results:For the virus spike-in samples, mVseq's analytical sensitivity and dynamic range were similar to quantitative PCR (qPCR). In clinical specimens, mVseq showed substantial agreement with single-target qPCR (92%; k statistic, 0.77; 259 of 282 viral tests); however, clinical sensitivity was reduced (81%), ranging from 62% to 100% for specific viruses. In 12 of the 47 patients tested, mVseq identified previously unknown BK virus, human herpesvirus-7, and Epstein-Barr virus infections that were confirmed by qPCR. Conclusions:Our results reveal factors that can influence clinical sensitivity, such as high levels of host DNA background and loss of detection in coinfections when 1 virus was at much higher concentration than the others. The mVseq assay is flexible and scalable to incorporate RNA viruses, emerging viruses of interest, and other pathogens important in transplant recipients.

SUBMITTER: Tan SK

PROVIDER: S-EPMC6594177 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Transplant Virus Detection Using Multiplex Targeted Sequencing.

Tan Susanna K SK Shen Peidong P Lefterova Martina I MI Sahoo Malaya K MK Fung Eula E Odegaard Justin I JI Davis Ronald W RW Pinsky Benjamin A BA Scharfe Curt C

The journal of applied laboratory medicine 20180301 5

<h4>Background</h4>Viral infections are a major cause of complications and death in solid organ and hematopoietic cell transplantation.<h4>Methods</h4>We developed a multiplex viral sequencing assay (mVseq) to simultaneously detect 20 transplant-relevant DNA viruses from small clinical samples. The assay uses a single-tube multiplex PCR to amplify highly conserved virus genomic regions without the need for previous virus enrichment or host nucleic acid subtraction. Multiplex sample sequencing wa ...[more]

PMID: 31245786

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Project description:Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R²?=?0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R²?=?0.97-0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R²??=?0.96) and whole transcriptome RNA-sequencing following "traditional" library preparation using Illumina NGS kits (R²?=?0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 10?-fold range was reduced more than 10,000-fold, from 2.3×10? to 1.4×10? sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.

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Transplant Virus Detection Using Multiplex Targeted Sequencing.

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Transplant Virus Detection Using Multiplex Targeted Sequencing.

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