• 대한임상검사과학회지
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• 제41권1호
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• pp.24-30
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• 2009

Rifampicin (RIF) and isoniazid (INH) are the most important drug for the treatment of Mycobacterium tuberculosis. Mutations correlated to rifampicin and isoniazid-resistance have been detected in rpoB gene and katG gene, respectively. Of the rifampicin-resistant isolates, 90% showed mutations in rpoB gene at codon 507 to 533. Isoniazid-resistant isolates analysed had a mutation in katG at codon 315. The aim of this study is to develop a pyrosequencing-based approach for rapid detection of ripampin or isoniazid resistant M. tuberculosis based on characterization of all possible mutation in the target region. For this study, the DNA selected from 35 cases of MTB PCR positive clinical sample such as bronchial washing, sputum, and pleural fluid. RIF or INH resistant was analyzed by pyrosequencing data of rpoB and katG gene. 28 (80%) and 7 (20%) of 35 MTB PCR positive DNAs were occured rifampicin-sensitivity and resistant, respectively. For INH, 30 (85.7%) and 5 (14.5%) cases were detected isoniazid-sensitivity and resistant, respectively. When pyrosequencing analysis was compared with ABI sequencing analysis, both analysis were presented same result, but pyrosequencing analysis was more rapid than ABI sequencing analysis. In conclusion, we found that pyrosequencing technology offers high accuracy, specificity, short turn around time and a high throughput in detection of rifampicin or isoniazid resistance in M. tuberculosis.

• 미생물학회지
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• 제49권2호
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• pp.150-155
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• 2013

Pyrosequencing을 이용하여 영산강 유역의 세균 군집 현황을 조사하였다. 영산강 본류 및 지천 14개 지점을 선정하여 5-7월동안 시료채취를 하였다. 총 42개의 시료를 가지고 총 989,380 reads을 얻었으며 taxonomic 분류와 OTU 분포도 분석을 실시하였다. Phylum 계층의 세균 구조 결과를 통해서 지천의 특성, 토지 이용, 시기적인 요인 등 환경적 요인에 따라 세균 군집 구조가 민감하게 변하는 경향을 나타내었다. 또한, OTU 분석을 근거한 오염원 추적기법(microbial source tracking; MST)을 통해 분변오염을 추적해보았다. Firmicutes가 가장 수질에 영향을 주는 taxa로 관찰되었다. 본 연구를 통해서, 현재 사용하는 수질 지표, BOD, pH 등에 더하여 pyrosequencing을 이용한 세균군집 조사 결과가 보다 효율적인 하천의 물관리 정책을 위해서 유용한 정보가 될 수 있을 것으로 기대한다.

• 한국미생물·생명공학회지
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• 제39권4호
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• pp.374-381
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• 2011

미생물과의 상호작용을 통하여 토양의 특성을 변화시킬 수 있는 지렁이 Eisenia fetida의 장내미생물 군집을 조사하기 위하여, 배양방법과 비배양방법인 DGGE와 pyrosequencing을 이용하여 8주와 16주 사육 지렁이의 장내미생물 군집을 분석하였다. 배양방법에서는 L. fusiformis(51%), B. cereus(30%), E. aerogenes(21%), 그리고 L. sphaericus (15%) 등이 우점미생물로 확인되었다. DGGE 분석에서는 B. cereus(15.1%), Enterobacter sp.(13.6%), uncultured bacterium (13.1%), 그리고 B. stearothermophilus(7.8%)가 우점미생물로 확인되었다. Pyrosequencing 분석에서는 Microbacterium soli(26%), B. cereus(10%), M. esteraromaticum(6%), 그리고 Frigoribacterium sp.(6%)가 우점미생물로 확인되었다. 그외에도 Aeromonas sp., Pseudomonas sp., Borrelia sp., Cellulosimicrobium sp., Klebsiella sp., and Leifsonia sp. 등의 미생물도 확인이 되었으며, 비배양 방법을 이용한 장내 미생물 군집 조사는 배양이 불가능한 미생물을 확인할 수 있을 뿐만 아니라 더 다양한 미생물 군집도 확인할 수 있었다.

• Journal of Korean Neurosurgical Society
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• 제59권1호
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• pp.26-36
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• 2016

Objective : This study investigated whether pyrosequencing can be used to determine the methylation status of the MGMT promoter as a clinical biomarker using relatively old archival tissue samples of glioblastoma. We also examined other prognostic factors for survival of glioblastoma patients. Methods : The available study set included formalin-fixed paraffin-embedded (FFPE) tissue from 104 patients at two institutes from 1997 to 2012, all of which were diagnosed histopathologically as glioblastoma. Clinicopathologic data were collected by review of medical records. For pyrosequencing analysis, the PyroMark Q96 CpG MGMT kit (Qiagen, Hilden, Germany) was used to detect the level of methylation at exon 1 positions 17-39 of the MGMT gene, which contains 5 CpGs. Results : Methylation of the MGMT promoter was detected in 43 (41.3%) of 104 samples. The average percentage methylation was $14.0{\pm}16.8%$ overall and $39.0{\pm}14.7%$ for methylated cases. There was no significant pattern of linear increase or decrease according to the age of the FFPE block (p=0.687). In multivariate analysis, age, performance status, extent of surgery, method of adjuvant therapy, and methylation status estimated by pyrosequencing were independently associated with overall survival. Additionally, patients with a high level of methylation survived longer than those with low methylation (p=0.016). Conclusion : In this study, the status and extent of methylation of the MGMT promoter analyzed by pyrosequencing were associated with overall survival in glioblastoma patients. Pyrosequencing is a quantitative method that overcomes the problems of MSP and a simple technique for accurate analysis of DNA sequences.

• 대한한의학회지
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• 제25권4호
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• pp.147-151
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• 2004

Objective : Angelica species are some of the most medicinally important materials in Oriental medicine. This study was performed to determine if Angelica species could be identified by genetic analysis and to verify Pyrosequencing analyses, which were used to assess genetic variation. Methods : The DNAs of Angelica acutiloba, Angelica gigas and Angelica sinensis were extracted. We have investigated the typing of single-base variations of Angelica species in DNA by using Pyrosequencing. Results : Angelica gigas showed a different pattern compared with Angelica acutiloba and Angelica sinensis. The peak of Angelica gigas was very weak in the second C nucleotide base compared with that of the others. The peak of Angelica acutiloba was present in the fourth C nucleotide base compared with that of the others. From these results we verified that our Angelica species-specific sequencing primer was well designed. Conclusion : Pyrosequencing analysis might be able to provide the identification of the Angelica species.

• Genomics & Informatics
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• 제6권2호
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• pp.87-90
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• 2008

During the last four years, the pyrosequencing-based 454 platform has rapidly displaced the traditional Sanger sequencing method due to its high throughput and cost effectiveness. Meanwhile, the Sanger sequencing methodology still provides the longest reads, and paired-end sequencing that is based on that chemistry offers an opportunity to ensure accurate assembly results. In this report, we describe an optimized approach for hybrid de novo genome assembly using pyrosequencing data and varying amounts of Sanger-type reads. 454 platform-derived contigs can be used as single non-breakable virtual reads or converted to simpler contigs that consist of editable, overlapping pseudoreads. These modified contigs maintain their integrity at the first jumpstarting assembly stage and are edited by fragmenting and rejoining. Pre-existing assembly software then can be applied for mixed assembly with 454-derived data and Sanger reads. An effective method for identifying genomic differences between reference and sample sequences in whole-genome resequencing procedures also is suggested.

• 대한임상검사과학회지
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• 제47권1호
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• pp.17-23
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• 2015

A fine needle aspiration biopsy (FNAB) is the primary means of distinguishing benign from malignant in thyroid nodules. However, between 10 and 30% of the FNABs of thyroid nodules are diagnosed as 'indeterminate'. A molecular method is needed to reduce unnecessary surgery in this group. In Korea, most thyroid cancer is classic papillary type and BRAFV600E mutation is highly prevalent. Thus, this study compared the pyrosequencing method with the conventional direct DNA sequencing and PCR-RFLP analysis and investigated the evaluation of preoperative BRAFV600E mutation analysis as an adjunct diagnostic method with routine FNABs. Sixty-five (78.3%) of 83 histopathologically diagnosed malignant nodule revealed positive BRAFV600E mutation on pyrosequencing analysis. In detail, 65 (83.8%) of 78 papillary thyroid carcinomas sample showed positive BRAFV600E mutation. None of 29 benign nodules had in pyrodequencing, direct DNA sequencing and PCR-RFLP. Out of 31 thyroid nodules classified as 'indeterminate' on cytological examination preoperatively, 28 cases turned out to be malignant: 24 papillary thyroid carcinomas. Among that, 16 (66.7%) classic papillary thyroid carcinomas had BRAFV600E mutation. Among 65 papillary thyroid carcinomas with positive BRAFV600E mutation detected by pyrosequencing analysis, each 3 cases and 5 cases did not show BRAFV600E mutation by direct DNA sequencing and PCR-RFLP analysis. Therefore, pyrosequencing was superior to direct DNA sequencing and PCR-RFLP in detecting the BRAFV600E mutation of thyroid nodules (p =0.027). Detecting BRAFV600E mutation by pyrosequencing was more sensitivity, faster than direct DNA sequencing or PCR-RFLP.

• 미생물학회지
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• 제46권4호
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• pp.352-358
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• 2010

서로 다른 처리공정으로 운영되는 4개의 하수시설을 대상으로 16S rRNA 유전자 기반의 pyrosequencing을 이용해 활성슬러지 하수처리 생물반응기의 세균군집구조를 분석하였다. 활성 슬러지에는 Rhodocyclales, Burkholderiales, Sphingobacteriales, Myxococcales, Xanthomonadales, Acidobacteria group 4, Anaerolineales, Methylococcales, Nitrospirales, Planctomycetales 목에 속하는 염기서열이 전체의 54-68%를 차지해, 소수의 세균 분류군이 활성슬러지 세균군집의 대부분을 차지하고 있었다. 이들 소수 세균 분류군의 조성은 처리장별로 차이가 있었으며, 하수처리장의 운전조건 및 환경조건에 영향을 받는 것으로 추측되었다. 또한, 활성슬러지는 매우 다양한 세균 종을 가지는 것으로 관찰되었는데(Chao1 richness estimate: 1,374-2,902 operational taxonomic units), 대부분의 다양성은 희귀 종에 기인한 것으로 나타났다. 특히, 분리막으로 운영되는 하수처리시설에서 높은 다양성을 나타내었는데, 처리공정이 매우 긴 고형물체류시간으로 운영되어 느리게 성장하는 다양한 세균이 서식하는데 용이하기 때문인 것으로 판단되었다. High-throughput pyrosequencing 기술을 이용하여 활성슬러지 세균군집을 처리장별로 비교 분석한 본 연구는 향후 활성슬러지 미생물의 생태학적 특성을 보다 잘 이해하고 하수처리공정을 개선하는데 도움이 될 것이다.

• 한국토양비료학회지
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• 제45권6호
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• pp.1073-1085
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• 2012

Soil microbial communities are immensely diverse and complex with respect to species richness and community size. These communities play essential roles in agricultural soil because they are responsible for most of the nutrient cycles in the soil and influence the plant diversity and productivity. However, the majority of these microbes remain uncharacterized because of poor culturability. Next-generation sequencing techniques have revolutionized many areas of biology by providing cheaper and faster alternatives to Sanger sequencing. Among them, amplicon pyrosequencing is a powerful tool developed by 454 Life Sciences for assessing the diversity of complex microbial communities by sequencing PCR products or amplicons. This review summarizes the current opinions in amplicon sequencing of soil microbial communities, and provides practical guidance and advice on sequence quality control, aligning, clustering, OTU- and taxon-based analysis. The last section of this article includes a few representative studies conducted using amplicon pyrosequencing.

• Journal of Microbiology and Biotechnology
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• 제25권1호
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• pp.1-10
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• 2015

The bacterial diversity of 10 marine sponges belonging to the species Cliona celata, an unidentified Cliona species, Haliclona cinerea, Halichondria okadai, Hymeniacidon sinapium, Lissodendoryx isodictyalis, Penares incrustans, Spirastrella abata, and Spirastrella panis collected from Jeju Island and Chuja Island was investigated using amplicon pyrosequencing of the 16S rRNA genes. The microbial diversity of these sponges has as of yet rarely or never been investigated. All sponges, except Cliona celata, Lissodendoryx isodictyalis, and Penares incrustans, showed simple bacterial diversity, in which one or two bacterial OTUs occupied more than 50% of the pyrosequencing reads and their OTU rank abundance curves saturated quickly. Most of the predominant OTUs belonged to Alpha-, Beta-, or Gammaproteobacteria. Some of the OTUs from the sponges with low diversity were distantly (88%~89%) or moderately (93%~97%) related to known sequences in the GenBank nucleotide database. Phylogenetic analysis showed that many of the representative sequences of the OTUs were related to the sequences originating from sponges and corals, and formed sponge-specific or -related clades. The marine sponges investigated herein harbored unexplored bacterial diversity, and further studies should be done to understand the microbes present in sponges.