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Insights into bacterioplankton community structure from Sundarbans mangrove ecoregion using Sanger and Illumina MiSeq sequencing approaches: A comparative analysis.

ABSTRACT: Next generation sequencing using platforms such as Illumina MiSeq provides a deeper insight into the structure and function of bacterioplankton communities in coastal ecosystems compared to traditional molecular techniques such as clone library approach which incorporates Sanger sequencing. In this study, structure of bacterioplankton communities was investigated from two stations of Sundarbans mangrove ecoregion using both Sanger and Illumina MiSeq sequencing approaches. The Illumina MiSeq data is available under the BioProject ID PRJNA35180 and Sanger sequencing data under accession numbers KX014101-KX014140 (Stn1) and KX014372-KX014410 (Stn3). Proteobacteria-, Firmicutes- and Bacteroidetes-like sequences retrieved from both approaches appeared to be abundant in the studied ecosystem. The Illumina MiSeq data (2.1 GB) provided a deeper insight into the structure of bacterioplankton communities and revealed the presence of bacterial phyla such as Actinobacteria, Cyanobacteria, Tenericutes, Verrucomicrobia which were not recovered based on Sanger sequencing. A comparative analysis of bacterioplankton communities from both stations highlighted the presence of genera that appear in both stations and genera that occur exclusively in either station. However, both the Sanger sequencing and Illumina MiSeq data were coherent at broader taxonomic levels. Pseudomonas, Devosia, Hyphomonas and Erythrobacter-like sequences were the abundant bacterial genera found in the studied ecosystem. Both the sequencing methods showed broad coherence although as expected the Illumina MiSeq data helped identify rarer bacterioplankton groups and also showed the presence of unassigned OTUs indicating possible presence of novel bacterioplankton from the studied mangrove ecosystem.

SUBMITTER: Ghosh A

PROVIDER: S-EPMC5133466 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Insights into bacterioplankton community structure from Sundarbans mangrove ecoregion using Sanger and Illumina MiSeq sequencing approaches: A comparative analysis.

Ghosh Anwesha A Bhadury Punyasloke P

Genomics data 20161124

Next generation sequencing using platforms such as Illumina MiSeq provides a deeper insight into the structure and function of bacterioplankton communities in coastal ecosystems compared to traditional molecular techniques such as clone library approach which incorporates Sanger sequencing. In this study, structure of bacterioplankton communities was investigated from two stations of Sundarbans mangrove ecoregion using both Sanger and Illumina MiSeq sequencing approaches. The Illumina MiSeq data ...[more]

PMID: 27942457

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Project description:BACKGROUND:Anthropogenic perturbations have strong impact on water quality and ecological health of mangrove areas of Indian Sundarbans. Diversity in microbial community composition is important causes for maintaining the health of the mangrove ecosystem. However, microbial communities of estuarine water in Indian Sundarbans mangrove areas and environmental determinants that contribute to those communities were seldom studied. METHODS:Nevertheless, this study attempted first to report bacterial and archaeal communities simultaneously in the water from Matla River and Thakuran River of Maipith coastal areas more accurately using 16S rRNA gene-based amplicon approaches. Attempt also been made to assess the capability of the environmental parameters for explaining the variation in microbial community composition. RESULTS:Our investigation indicates the dominancy of halophilic marine bacteria from families Flavobacteriaceae and OM1 clade in the water with lower nutrient load collected from costal regions of a small Island of Sundarban Mangroves (ISM). At higher eutrophic conditions, changes in bacterial communities in Open Marine Water (OMW) were detected, where some of the marine hydrocarbons degrading bacteria under families Oceanospirillaceae and Spongiibacteraceae were dominated. While most abundant bacterial family Rhodobacteracea almost equally (18% of the total community) dominated in both sites. Minor variation in the composition of archaeal community was also observed between OMW and ISM. Redundancy analysis indicates a combination of total nitrogen and dissolved inorganic nutrients for OMW and for ISM, salinity and total nitrogen was responsible for explaining the changes in their respective microbial community composition. CONCLUSIONS:Our study contributes the first conclusive overview on how do multiple environmental/anthropogenic stressors (salinity, pollution, eutrophication, land-use) affect the Sundarban estuary water and consequently the microbial communities in concert. However, systematic approaches with more samples for evaluating the effect of environmental pollutions on mangrove microbial communities are recommended.

Project description:The chloroplast genome is important for plant development and plant evolution. Nelumbo nucifera is one member of relict plants surviving from the late Cretaceous. Recently, a new sequencing platform PacBio RS II, known as 'SMRT (Single Molecule, Real-Time) sequencing', has been developed. Using the SMRT sequencing to investigate the chloroplast genome of N. nucifera will help to elucidate the plastid evolution of basal eudicots.The sizes of the de novo assembled complete chloroplast genome of N. nucifera were 163,307 bp, 163,747 bp and 163,600 bp with average depths of coverage of 7×, 712× and 105× sequenced by Sanger, Illumina MiSeq and PacBio RS II, respectively. The precise chloroplast genome of N. nucifera was obtained from PacBio RS II data proofread by Illumina MiSeq reads, with a quadripartite structure containing a large single copy region (91,846 bp) and a small single copy region (19,626 bp) separated by two inverted repeat regions (26,064 bp). The genome contains 113 different genes, including four distinct rRNAs, 30 distinct tRNAs and 79 distinct peptide-coding genes. A phylogenetic analysis of 133 taxa from 56 orders indicated that Nelumbo with an age of 177 million years is a sister clade to Platanus, which belongs to the basal eudicots. Basal eudicots began to emerge during the early Jurassic with estimated divergence times at 197 million years using MCMCTree. IR expansions/contractions within the basal eudicots seem to have occurred independently.Because of long reads and lack of bias in coverage of AT-rich regions, PacBio RS II showed a great promise for highly accurate 'finished' genomes, especially for a de novo assembly of genomes. N. nucifera is one member of basal eudicots, however, evolutionary analyses of IR structural variations of N. nucifera and other basal eudicots suggested that IR expansions/contractions occurred independently in these basal eudicots or were caused by independent insertions and deletions. The precise chloroplast genome of N. nucifera will present new information for structural variation of chloroplast genomes and provide new insight into the evolution of basal eudicots at the primary sequence and structural level.

Project description:Illumina's MiSeq has become the dominant platform for gene amplicon sequencing in microbial ecology studies; however, various technical concerns, such as reproducibility, still exist. To assess reproducibility, 16S rRNA gene amplicons from 18 soil samples of a reciprocal transplantation experiment were sequenced on an Illumina MiSeq. The V4 region of 16S rRNA gene from each sample was sequenced in triplicate with each replicate having a unique barcode. The average OTU overlap, without considering sequence abundance, at a rarefaction level of 10,323 sequences was 33.4±2.1% and 20.2±1.7% between two and among three technical replicates, respectively. When OTU sequence abundance was considered, the average sequence abundance weighted OTU overlap was 85.6±1.6% and 81.2±2.1% for two and three replicates, respectively. Removing singletons significantly increased the overlap for both (~1-3%, p<0.001). Increasing the sequencing depth to 160,000 reads by deep sequencing increased OTU overlap both when sequence abundance was considered (95%) and when not (44%). However, if singletons were not removed the overlap between two technical replicates (not considering sequence abundance) plateaus at 39% with 30,000 sequences. Diversity measures were not affected by the low overlap as α-diversities were similar among technical replicates while β-diversities (Bray-Curtis) were much smaller among technical replicates than among treatment replicates (e.g., 0.269 vs. 0.374). Higher diversity coverage, but lower OTU overlap, was observed when replicates were sequenced in separate runs. Detrended correspondence analysis indicated that while there was considerable variation among technical replicates, the reproducibility was sufficient for detecting treatment effects for the samples examined. These results suggest that although there is variation among technical replicates, amplicon sequencing on MiSeq is useful for analyzing microbial community structure if used appropriately and with caution. For example, including technical replicates, removing spurious sequences and unrepresentative OTUs, using a clustering method with a high stringency for OTU generation, estimating treatment effects at higher taxonomic levels, and adapting the unique molecular identifier (UMI) and other newly developed methods to lower PCR and sequencing error and to identify true low abundance rare species all can increase reproducibility.

Dataset Information

Insights into bacterioplankton community structure from Sundarbans mangrove ecoregion using Sanger and Illumina MiSeq sequencing approaches: A comparative analysis.

Publications

Insights into bacterioplankton community structure from Sundarbans mangrove ecoregion using Sanger and Illumina MiSeq sequencing approaches: A comparative analysis.

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