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Development and bias assessment of a method for targeted metagenomic sequencing of marine cyanobacteria.

ABSTRACT: Prochlorococcus and Synechococcus are the most abundant photosynthetic organisms in oligotrophic waters and responsible for a significant percentage of the earth's primary production. Here we developed a method for metagenomic sequencing of sorted Prochlorococcus and Synechococcus populations using a transposon-based library preparation technique. First, we observed that the cell lysis technique and associated amount of input DNA had an important role in determining the DNA library quality. Second, we found that our transposon-based method provided a more even coverage distribution and matched more sequences of a reference genome than multiple displacement amplification, a commonly used method for metagenomic sequencing. We then demonstrated the method on Prochlorococcus and Synechococcus field populations from the Sargasso Sea and California Current isolated by flow cytometric sorting and found clear environmentally related differences in ecotype distributions and gene abundances. In addition, we saw a significant correspondence between metagenomic libraries sequenced with our technique and regular sequencing of bulk DNA. Our results show that this targeted method is a viable replacement for regular metagenomic approaches and will be useful for identifying the biogeography and genome content of specific marine cyanobacterial populations.

SUBMITTER: Batmalle CS

PROVIDER: S-EPMC3911227 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Development and bias assessment of a method for targeted metagenomic sequencing of marine cyanobacteria.

Batmalle Cécilia S CS Chiang Hsin-I HI Zhang Kun K Lomas Michael W MW Martiny Adam C AC

Applied and environmental microbiology 20131202 3

Prochlorococcus and Synechococcus are the most abundant photosynthetic organisms in oligotrophic waters and responsible for a significant percentage of the earth's primary production. Here we developed a method for metagenomic sequencing of sorted Prochlorococcus and Synechococcus populations using a transposon-based library preparation technique. First, we observed that the cell lysis technique and associated amount of input DNA had an important role in determining the DNA library quality. Seco ...[more]

PMID: 24296495

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Project description:Current diagnostics and clinical management strategies for combat wounds are based on decisions made by expert clinicians. However, even in the hands of experienced surgeons, wounds from combat injuries can exhibit failed healing and complications related to limitations in the rapid and comprehensive generation of diagnostic information. Previous studies have demonstrated the possible use of genomic sequencing approaches to detect microbial signatures involved in combat casualty care. While effective, whole metagenome sequencing is limited by the depth required to confidently detect all relevant signatures. To address this, we developed a targeted capture sequencing panel to detect microbial signatures relevant to wound healing. These targets include known microbial nosocomial pathogens, wound colonizers, and genes involved in virulence and antimicrobial resistance. A bioinformatics pipeline was built to identify genomic regions of interest and over 8,000 oligonucleotide probes were designed for capture. The panel was synthesized and validated using control reference genomes in human background and on wound-effluent samples from a cohort of combat-injured U.S. service members. Our panel was sensitive against wound-colonizing species, Acinetobacter baumannii and Pseudomonas aeruginosa, and was specific in detecting corresponding virulence and antimicrobial-resistance genes as well as other pathogenic species present in microflora mixtures. Random forest feature permutation confirmed the prevalence of Acinetobacter and Pseudomonas in critically colonized wounds and wounds that failed to heal, respectively. Our results demonstrate the capability of targeted sequencing tools and analysis platforms to profile and deliver information on pathogenic factors influencing wound progression, thereby guiding therapeutic intervention. IMPORTANCE Microbial contamination in combat wounds can lead to opportunistic infections and adverse outcomes. However, current microbiological detection has a limited ability to capture microbial functional genes. This work describes the application of targeted metagenomic sequencing to profile wound bioburden and capture relevant wound-associated signatures for clinical utility. Ultimately, the ability to detect such signatures will help guide clinical decisions regarding wound care and management and aid in the prediction of wound outcomes.

Project description:Metagenomics is increasingly used to describe microbial communities in biological specimens. Ideally, the steps involved in the processing of the biological specimens should not change the microbiome composition in a way that it could lead to false interpretations of inferred microbial community composition. Common steps in sample preparation include sample collection, storage, DNA isolation, library preparation, and DNA sequencing. Here, we assess the effect of three library preparation kits and two DNA sequencing platforms. Of the library preparation kits, one involved a PCR step (Nextera), and two were PCR free (NEXTflex and KAPA). We sequenced the libraries on Illumina HiSeq and NextSeq platforms. As example microbiomes, two pig fecal samples and two sewage samples of which aliquots were stored at different storage conditions (immediate processing and storage at -80°C) were assessed. All DNA isolations were performed in duplicate, totaling 80 samples, excluding controls. We found that both library preparation and sequencing platform had systematic effects on the inferred microbial community composition. The different sequencing platforms introduced more variation than library preparation and freezing the samples. The results highlight that all sample processing steps need to be considered when comparing studies. Standardization of sample processing is key to generating comparable data within a study, and comparisons of differently generated data, such as in a meta-analysis, should be performed cautiously. IMPORTANCE Previous research has reported effects of sample storage conditions and DNA isolation procedures on metagenomics-based microbiome composition; however, the effect of library preparation and DNA sequencing in metagenomics has not been thoroughly assessed. Here, we provide evidence that library preparation and sequencing platform introduce systematic biases in the metagenomic-based characterization of microbial communities. These findings suggest that library preparation and sequencing are important parameters to keep consistent when aiming to detect small changes in microbiome community structure. Overall, we recommend that all samples in a microbiome study are processed in the same way to limit unwanted variations that could lead to false conclusions. Furthermore, if we are to obtain a more holistic insight from microbiome data generated around the world, we will need to provide more detailed sample metadata, including information about the different sample processing procedures, together with the DNA sequencing data at the public repositories.

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Dataset Information

Development and bias assessment of a method for targeted metagenomic sequencing of marine cyanobacteria.

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Development and bias assessment of a method for targeted metagenomic sequencing of marine cyanobacteria.

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