Dataset Information

Characterization of Intestinal Microbiomes of Hirschsprung's Disease Patients with or without Enterocolitis Using Illumina-MiSeq High-Throughput Sequencing.

ABSTRACT: Hirschsprung-associated enterocolitis (HAEC) is a life-threatening complication of Hirschsprung's disease (HD). Although the pathological mechanisms are still unclear, studies have shown that HAEC has a close relationship with the disturbance of intestinal microbiota. This study aimed to investigate the characteristics of the intestinal microbiome of HD patients with or without enterocolitis. During routine or emergency surgery, we collected 35 intestinal content samples from five patients with HAEC and eight HD patients, including three HD patients with a history of enterocolitis who were in a HAEC remission (HAEC-R) phase. Using Illumina-MiSeq high-throughput sequencing, we sequenced the V4 region of bacterial 16S rRNA, and operational taxonomic units (OTUs) were defined by 97% sequence similarity. Principal coordinate analysis (PCoA) of weighted UniFrac distances was performed to evaluate the diversity of each intestinal microbiome sample. The microbiota differed significantly between the HD patients (characterized by the prevalence of Bacteroidetes) and HAEC patients (characterized by the prevalence of Proteobacteria), while the microbiota of the HAEC-R patients was more similar to that of the HAEC patients. We also observed that the specimens from different intestinal sites of each HD patient differed significantly, while the specimens from different intestinal sites of each HAEC and HAEC-R patient were more similar. In conclusion, the microbiome pattern of the HAEC-R patients was more similar to that of the HAEC patients than to that of the HD patients. The HD patients had a relatively distinct, more stable community than the HAEC and HAEC-R patients, suggesting that enterocolitis may either be caused by or result in a disruption of the patient's uniquely adapted intestinal flora. The intestinal microbiota associated with enterocolitis may persist following symptom resolution and can be implicated in the symptom recurrence.

SUBMITTER: Li Y

PROVIDER: S-EPMC5014423 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

Characterization of Intestinal Microbiomes of Hirschsprung's Disease Patients with or without Enterocolitis Using Illumina-MiSeq High-Throughput Sequencing.

Li Yuqing Y Poroyko Valeriy V Yan Zhilong Z Pan Liya L Feng Yi Y Zhao Peihua P Xie Zhoulonglong Z Hong Li L

PloS one 20160907 9

Hirschsprung-associated enterocolitis (HAEC) is a life-threatening complication of Hirschsprung's disease (HD). Although the pathological mechanisms are still unclear, studies have shown that HAEC has a close relationship with the disturbance of intestinal microbiota. This study aimed to investigate the characteristics of the intestinal microbiome of HD patients with or without enterocolitis. During routine or emergency surgery, we collected 35 intestinal content samples from five patients with ...[more]

PMID: 27603009

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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

Characterization of Intestinal Microbiomes of Hirschsprung's Disease Patients with or without Enterocolitis Using Illumina-MiSeq High-Throughput Sequencing.

Publications

Characterization of Intestinal Microbiomes of Hirschsprung's Disease Patients with or without Enterocolitis Using Illumina-MiSeq High-Throughput Sequencing.

Similar Datasets

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