Dataset Information

MultiplexSSR: A pipeline for developing multiplex SSR-PCR assays from resequencing data.

ABSTRACT: Next-generation sequencing has greatly promoted the investigation of single nucleotide polymorphisms, while studies of simple sequence repeats are sharply decreasing. However, simple sequence repeats still present some advantages in conservation genetics. In this study, an end-to-end pipeline referred to as MultiplexSSR was established to develop multiplex PCR assays in batches with highly polymorphic simple sequence repeats for capillary platforms from resequencing data. The distribution of single sequence repeats in the genome, the error profiles of genotypes and allelotypes, and the increase in the allele length range depending on the number of individuals were investigated. A total of 98% of single sequence repeats presented lengths of less than 100 bp. The error rate of the genotyping and allelotyping of dimeric patterns was ten times higher than those for other patterns. The error rate of allelotyping was less than that of genotyping. The allele length range reached approximate saturation with 10 individuals. This pipeline uses allele numbers to select highly polymorphic loci, masks loci with variation, and applies in silico PCR to improve primer specificity. The application of the developed multiplex SSR-PCR assays validated the pipeline's robustness, showing higher polymorphism and stability for the developed simple sequence repeats and a lower cost for genotyping and providing low-depth resequencing data from less than a dozen individuals for the development of markers. This pipeline fills the gap between next-generation sequencing and multiplex SSR-PCR.

SUBMITTER: Guo L

PROVIDER: S-EPMC7083706 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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MultiplexSSR: A pipeline for developing multiplex SSR-PCR assays from resequencing data.

Guo Liang L Yang Quan Q Yang Jing-Wen JW Zhang Nan N Liu Bao-Suo BS Zhu Ke-Cheng KC Guo Hua-Yang HY Jiang Shi-Gui SG Zhang Dian-Chang DC

Ecology and evolution 20200304 6

Next-generation sequencing has greatly promoted the investigation of single nucleotide polymorphisms, while studies of simple sequence repeats are sharply decreasing. However, simple sequence repeats still present some advantages in conservation genetics. In this study, an end-to-end pipeline referred to as MultiplexSSR was established to develop multiplex PCR assays in batches with highly polymorphic simple sequence repeats for capillary platforms from resequencing data. The distribution of sin ...[more]

PMID: 32211176

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Project description:A large number of viral and bacterial organisms are responsible for community-acquired pneumonia (CAP) which contributes to substantial burden on health management. A new resequencing microarray (RPM-IVDC1) associated with targeted multiplex PCR was recently developed and validated for multiple respiratory viruses detection and discrimination. In this study, we evaluated the capability of RPM-IVDC1 for simultaneous identification of multiple viral and bacterial organisms. The nasopharyngeal aspirates (NPAs) of 110 consecutive CAP patients, aged from 1 month to 96 years old, were collected from five distinct general hospitals in Beijing during 1-year period. The samples were subjected to the RPM-IVDC1 established protocol as compared to a real-time PCR (qRT-PCR), which was used as standard. The results of virus detection were consistent with those previously described. A total of 37 of Streptococcus pneumoniae, 14 of Haemophilus influenzae, 10 of Mycoplasma pneumoniae, two of Klebsiella pneumoniae and one of Moraxella catarrhalis were detected by RPM-IVDC1. The sensitivities and specificities were compared with those of qRT-PCR for S. pneumoniae (100, 100%, respectively), H. influenzae (92.3, 97.9%, respectively), M. pneumoniae (69.2, 99.0%, respectively), K. pneumoniae (100, 100%, respectively), and M. catarrhalis (100, 100%, respectively). Additional 22 of Streptococcus spp., 24 of Haemophilus spp. and 16 of Neisseria spp. were identified. In addition, methicillin-resistant and carbapenemases allele were also found in nine of Staphylococcus spp. and one of K. pneumoniae, respectively. These results demonstrated the capability of RPM-IVDC1 for simultaneous detection of broad-spectrum respiratory pathogens in complex backgrounds and the advantage of accessing to the actual sequences, showing great potential use of epidemic outbreak investigation. The detection results should be carefully interpreted when introducing this technique in the clinical diagnostics.

Dataset Information

MultiplexSSR: A pipeline for developing multiplex SSR-PCR assays from resequencing data.

Publications

MultiplexSSR: A pipeline for developing multiplex SSR-PCR assays from resequencing data.

Similar Datasets

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