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Assembly methods for nanopore-based metagenomic sequencing: a comparative study.

ABSTRACT: Metagenomic sequencing has allowed for the recovery of previously unexplored microbial genomes. Whereas short-read sequencing platforms often result in highly fragmented metagenomes, nanopore-based sequencers could lead to more contiguous assemblies due to their potential to generate long reads. Nevertheless, there is a lack of updated and systematic studies evaluating the performance of different assembly tools on nanopore data. In this study, we have benchmarked the ability of different assemblers to reconstruct two different commercially-available mock communities that have been sequenced using Oxford Nanopore Technologies platforms. Among the tested tools, only metaFlye, Raven, and Canu performed well in all the datasets. These tools retrieved highly contiguous genomes (or even complete genomes) directly from the metagenomic data. Despite the intrinsic high error of nanopore sequencing, final assemblies reached high accuracy (~?99.5 to 99.8% of consensus accuracy). Polishing strategies demonstrated to be necessary for reducing the number of indels, and this had an impact on the prediction of biosynthetic gene clusters. Correction with high quality short reads did not always result in higher quality draft assemblies. Overall, nanopore metagenomic sequencing data-adapted to MinION's current output-proved sufficient for assembling and characterizing low-complexity microbial communities.

SUBMITTER: Latorre-Perez A

PROVIDER: S-EPMC7423617 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Assembly methods for nanopore-based metagenomic sequencing: a comparative study.

Latorre-Pérez Adriel A Villalba-Bermell Pascual P Pascual Javier J Vilanova Cristina C

Scientific reports 20200812 1

Metagenomic sequencing has allowed for the recovery of previously unexplored microbial genomes. Whereas short-read sequencing platforms often result in highly fragmented metagenomes, nanopore-based sequencers could lead to more contiguous assemblies due to their potential to generate long reads. Nevertheless, there is a lack of updated and systematic studies evaluating the performance of different assembly tools on nanopore data. In this study, we have benchmarked the ability of different assemb ...[more]

PMID: 32788623

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Project description:BACKGROUND:Prosthetic joint infections are clinically difficult to diagnose and treat. Previously, we demonstrated metagenomic sequencing on an Illumina MiSeq replicates the findings of current gold standard microbiological diagnostic techniques. Nanopore sequencing offers advantages in speed of detection over MiSeq. Here, we report a real-time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections but potentially useful for any microbial sequencing, and compare detection by direct-from-clinical-sample metagenomic nanopore sequencing with Illumina sequencing and standard microbiological diagnostic techniques. RESULTS:DNA was extracted from the sonication fluids of seven explanted orthopaedic devices, and additionally from two culture negative controls, and was sequenced on the Oxford Nanopore Technologies MinION platform. A specific analysis pipeline was assembled to overcome the challenges of identifying the true infecting pathogen, given high levels of host contamination and unavoidable background lab and kit contamination. The majority of DNA classified (>?90%) was host contamination and discarded. Using negative control filtering thresholds, the species identified corresponded with both routine microbiological diagnosis and MiSeq results. By analysing sequences in real time, causes of infection were robustly detected within minutes from initiation of sequencing. CONCLUSIONS:We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data and use of this pipeline to show initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. The high proportion of human DNA in prosthetic joint infection extracts prevents full genome analysis from complete coverage, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling. The nine samples sequenced in this pilot study have shown a proof of concept for sequencing and analysis that will enable us to investigate further sequencing to improve specificity and sensitivity.

Project description:Patients who were infected by the Human Immunodeficiency Virus (HIV) could have weakened immunity that is complicated by opportunistic infections, especially for Mycobacterium tuberculosis (MTB). Notably, the HIV-MTB co-infection will accelerate the course of disease progress and greatly increase the mortality of patients. Since the traditional diagnostic methods are time-consuming and have low sensitivity, we aim to investigate the performance of mNGS (metagenomic Next-Generation Sequencing) and mNPS (metagenomic NanoPore Sequencing) for the rapid diagnosis of tuberculosis in HIV-infected patients. The 122 HIV-infected patients were enrolled for the retrospective analysis. All of the patients underwent traditional microbiological tests, mNGS, and (or) mNPS tests. The clinical comprehensive diagnosis was used as the reference standard to compare the diagnostic performance of culture, mNGS, and mNPS on tuberculosis. We also investigate the diagnostic value of mNGS and mNPS on mixed-infection. Furthermore, the treatment adjustment directed by mNGS and mNPS was analyzed. Compared with the composite reference standard, the culture showed 42.6% clinical sensitivity and 100% specificity, and the OMT(other microbiological testing) had 38.9% sensitivity and 100% specificity. The mNGS had 58.6% clinical sensitivity and 96.8% specificity, and the mNPS had 68.0% clinical sensitivity and 100% specificity. The proportion of mixed-infection cases (88.9%) in the TB group was higher than those in the non-TB group (54.8%) and the mNGS and mNPS are more competitive on mixed-infection diagnosis compared with the traditional methods. Furthermore, there are 63 patients (69.2%) and 36 patients (63.2%) achieved effective treatment after receiving the detection of mNPS and mNGS, respectively. Our study indicated that mNPS and mNGS have high sensitivity and specificity for TB diagnosis compared with the traditional methods, and mNPS seems to have better diagnostic performance than mNGS. Moreover, mNGS and mNPS showed apparent advantages in detecting mixed infection. The mNPS and mNGS-directed medication adjustment have effective treatment outcomes for HIV-infected patients who have lower immunity.

Dataset Information

Assembly methods for nanopore-based metagenomic sequencing: a comparative study.

Publications

Assembly methods for nanopore-based metagenomic sequencing: a comparative study.

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