MicroPredict: predicting species-level taxonomic abundance of whole-shotgun metagenomic data using only 16S amplicon sequencing data

Jang Chloe Soohyun,Kim Hakin,Kim Donghyun,Han Buhm 한국유전학회 2024 Genes & Genomics Vol.46 No.6

Background The importance of the human microbiome in the analysis of various diseases is emerging. The two main methods used to profile the human microbiome are 16S rRNA gene sequencing (16S sequencing) and whole-genome shotgun sequencing (WGS). Owing to the full coverage of the genome in sequencing, WGS has multiple advantages over 16S sequencing, including higher taxonomic profiling resolution at the species-level and functional profiling analysis. However, 16S sequencing remains widely used because of its relatively low cost. Although WGS is the standard method for obtaining accurate species-level data, we found that 16S sequencing data contained rich information to predict high-resolution species-level abundances with reasonable accuracy. Objective In this study, we proposed MicroPredict, a method for accurately predicting WGS-comparable species-level abundance data using 16S taxonomic profile data. Methods We employed a mixed model using two key strategies: (1) modeling both sample- and species-specific information for predicting WGS abundances, and (2) accounting for the possible correlations among different species. Results We found that MicroPredict outperformed the other machine learning methods. Conclusion We expect that our approach will help researchers accurately approximate the species-level abundances of microbiome profiles in datasets for which only cost-effective 16S sequencing has been applied. Background The importance of the human microbiome in the analysis of various diseases is emerging. The two main methods used to profile the human microbiome are 16S rRNA gene sequencing (16S sequencing) and whole-genome shotgun sequencing (WGS). Owing to the full coverage of the genome in sequencing, WGS has multiple advantages over 16S sequencing, including higher taxonomic profiling resolution at the species-level and functional profiling analysis. However, 16S sequencing remains widely used because of its relatively low cost. Although WGS is the standard method for obtaining accurate species-level data, we found that 16S sequencing data contained rich information to predict high-resolution species-level abundances with reasonable accuracy. Objective In this study, we proposed MicroPredict, a method for accurately predicting WGS-comparable species-level abundance data using 16S taxonomic profile data. Methods We employed a mixed model using two key strategies: (1) modeling both sample- and species-specific information for predicting WGS abundances, and (2) accounting for the possible correlations among different species. Results We found that MicroPredict outperformed the other machine learning methods. Conclusion We expect that our approach will help researchers accurately approximate the species-level abundances of microbiome profiles in datasets for which only cost-effective 16S sequencing has been applied.

16S rRNA 유전자 염기서열 분석에 의해 확인된 Acinetobacter spp. 가성요로감염 유행

김수연,김진용,강지혜,박신영,이희승,박윤수,서일혜,조용균 대한감염학회 2007 감염과 화학요법 Vol.39 No.4

목적 : 본 연구는 일개 대학병원의 한 병동에서 16SrRNA 유전자 염기서열 분석을 통해 확인된 Acinetobacter spp. 가성요로감염 집단 발생에 대한 조사이다. 재료 및 방법 : 일개 대학병원의 일반병동에서 2005년 9월 23일부터 26일까지 5명의 환자에서 Bordetelta bronchiseptica 세균뇨가 동시에 분리되었다. 해당 환자들에 대한 입원 진료 기록을 확인하고, 이학적 검사를 시행하였고, 의료진 면담 등의 역학적 조사와 의심되는 전파원의 환경 감시배양을 시행하였다. 또한 다섯 균주들의 상동성 확인을 위해 pulsed field gel electrophoresis (PFGE)를 하였고, 정확한 균 동정을 위해 16S rRNA 유전자 염기서열 분석을 하였다. 결과 : VITEK system에 의해 B. bronchiseptica로 보고된 다섯 균주들은 거의 유사한 항생제 감수성을 가지고 있었다. 유행조사에서 요로감염의 증상이나 균혈증을 보인 환자는 없었고, 환경 감시배양에서 공통의 전파원은 증명되지 않았다. 또한 PFGE와 16S rRNA 유전자 염기서열분석에서 상동성을 가진 동일 Acinetobacter spp.로 확인되어 이에 의한 가성요로감염의 유행으로 결론지었다. 결론 : 역학적 조사와 함께 PFGE와 16s rRNA 유전자염기서열 분석과 같은 분자생물학적인 조사를 시행하는 것은 희귀한 균에 의한 병원감염 유행조사에 도움이 될 것이다. Background : Acinetobacter spp. is increasingly implicated in hospital-acquired infections. We experienced a pseudooutbreak of Bordetella bronchiseptica bacteriuria identified with biochemical tests, that was later identified as Acinetobacter spp. by using 16S rRNA gene sequence analysis. Materials and Methods : Five in-ward patients were found to have B. bronchiseptica bacteriuria without symptoms of urinary tract infection between September 23 and 26 of 2005. We conducted pulsed field gel electrophoresis (PFGE) of the bacteria and epidemiological investigation of this pseudooutbreak. In addition, 16S rRNA gene sequence analysis was performed for the verification of the strains. Results : All 5 isolates were identified as B. bronchiseptica with similar antibiogram by VITEK system. There was no evidence of any symptom or sign of urinary tract infection. The source of this pseudooutbreak was not detected even after performing environmental culture and interviews with healthcare workers. We could not get the appropriate results from the first PFGE with XbaI restriction enzyme. B. bronchiseptica is an unusual organism in human so we conducted 16S rRNA gene sequence analysis for verification. The analysis of 16S rRNA gene sequence with 5 isolates demonstrated 99-100% similarity to a sequence of Acinetobacter spp. (AU1523). According to the results of 16S rRNA gene sequence analysis, we performed the second PFGE with SmaI restriction enzyme, which showed indistinguishable pattern among the all 5 isolates. Conclusion : This investigation suggests that the combined method of 16s rRNA gene sequence analysis and PFGE would be helpful for investigation of outbreak caused by unusual organisms.

16S rRNA와 16S-23S rRNA Gene Sequence Analysis을 이용한 물김치와 동치미 유산균의 분리 동정

설수연,김태운,김성언,김해영 경희대학교식량자원개발연구소 2007 硏究論文集 Vol.26 No.1

This study was carried out to identify lactic acid bacteria in Mul-Kimchi and Dongchimi using by 16S rRNA and 16S-23S gene sequence analysis. Diluted Mul-Kimchi and Dongchimi soup were plated on the MRS agar media and colonies obtained were used for 16S rRNA and 16S-23S rRNA gene sequence analysis. Genomic DNAs were extracted from the isolated lactic acid bacteria and used as a template for PCR amplification. Universal primer sets based on the 16S rRNA and 16S-23S intergenic spacer region genes were used for amplification. From the sequencing of PCR products, Weissella kimchii, Weissella cibaria, Leuconostoc lactis, Leuconostoc citreum and Leuconostoc garlicum were identified.

Lactobacillus acidophilus ATCC 4356의 16S-23S rRNA intergenic spacer region sequence 특성과 균체 섭취에 의한 Salmonella enteritigis 감염시 쥐의 체중변화에 미치는 영향

배진성,윤영호 中央大學校 食糧資源硏究所 2002 食糧資源硏究所 論文集 Vol.14 No.1

Lactobacillus acidophilus의 16S-23S rRNA intergenic spacer region의 특성과 Lactobacillus spp. 급여에 의하여 Salmonella enteritidis 감염 이후 체중저하에 대한 보호효과를 주요 장기 중에 오염된 생균수 및 장기 중량 변화성향을 측정한 결과는 다음과 같다. Lactobacillus acidophilus의 16S-23S rRNA intergenic spacer region의 sequence를 측정하고 gene bank에 등록된 균주의 sequence와 비교한 결과 homology의 차이를 나타내었다 Lactobacillus spp. 급여 이후 Lactobacillus enteritidis를 challenge 이후 체중 변화는 4일 경과 후부터 체중 감소현상을 보였으며 체중 감소율이 가장 낮은 균주는 Lb. helveticus CU 631이었으며 균주간의 차이를 나타내었고, 간과 비장에 Salmonella enteritidis 오염 생균수는 Lactobacillus spp. 급여군이 대조군보다 낮은 성행을 보였고 간장 및 비장의 중량은 대조군보다 낮은 성향을 보였다. 16S-23S rRNA intergenic spacer region sequence and effects of Lactobacillus acidophilus ATCC 4356 against typical enteritis causing pathogen Salmonella enteritidis were determinded. Changes in the weight of body, spleen, liver and the contaminati on level of Salmonella enteritidis in the vital organ after challenge have been determined. The sequence of 16S-23S rRNA intergenic spacer region of Lactobacillus acidophilus could be utilized as a strain identification, those sequences comparison showed difference in homology. A rapid decline in body weight after seven days of challange with Salmonella enteritidis were observed in control mice, the protective effects from the decline in body weight by feeding Lactobacillus spp was most prominent in Lb. helveticus CU 631 and in Lb. acidophilus, more numerous viable number of Salmonella enteritidis in the tissues of spleen and liver and heavier average organ weight in control mice(unfed with Lactobacillus spp.) than lactobacillus spp. fed mice was observed.

Lactobacillus spp의 Salmonella enteritidis KU 101에 대한 보호 효과와 L. casei YIT 9018의 16S-23S rRNA Intergenic Spacer Region 염기배열 특성

배진성,윤영호 한국동물자원과학회 2003 한국축산학회지 Vol.45 No.3

Lactobacillus spp. 급여에 의하여 Salmonella enteritidis 감염에 의한 치사율의 저하 성향, 시험관내 억제활성, 장액 내의 총 IgA 농도변화와 Lactobacillus casei. 의 16S-23S rRNA intergenic spacer region의 sequence를 측정 비교한 결과는 다음과 같다. Lactobacillus spp. 5균주는 시험관내 Salmonella enteritidis 억제활성 측정 결과 모든 시험균주가 억제활성을 나타내었고 그 억제활성의 차이가 통계적인 유의성을 보였으며, 억제활성의 차이는 L. helveticus CU 631 > L. rhamnosus GG ATCC 4356 > L. casei YIT 9018 인 것으로 나타났다. Lactobacillus spp. 급여에 의하여 Salmonella enteritidis challenge에 의한 치사율 저하효과는 Lb. helveticus CU 631은대 대조구의 생존율 62%에 대하여 100%로서 가장 높은 치사율 저하효과를 나타내었고, L casei YIT 9018, L johnsonii C-4의 생존률이 70%와 50%를 나타내었다. Lactobacillus spp. 의 16S-23s rRNA intergenic spacer region의 sequence를 측정하고 gene bank에 등록된 균주의 sequence와 비교한 결과 homology의 차이를 나타내었다. In vivo protective and in vitro inhibitory activities of Lactobacillus casei YIT 9018. against typical enteritis causing Salmonella enteritidis KU101 and IgA level after challenge have been determined. In order to identify the strains of lactobacilli the sequences of 16S-23S rRNA intergenic spacer region were determined. All the test strains of Lactobacillus spp. inhibited Salmonella enteritidis, the intensity varied depending upon the species of lactobacilli. Effects on the survival rate of the mouse after challenge with Salmonella enteritidis KU101 on feeding Lactobacillus spp. have shown the highest survival rate in L. helveticus CU 631 followed by L. casei YIT 9018 and L. johnsonii C-4 and the lowest in control mice. The higher level of total Ig A concentration in the intestinal fluid of lactobacilli fed mice than control mice was observed. The sequences of 16S-23S rRNA intergenic spacer region of seven strains of Lactobacillus casei could be utilized as a strain identification, those sequences showed some degree of difference in homology.

유전자종 분석을 이용한 Acinetobacter의 균종 동정

이승호,이미경,박애자,최응상 대한임상병리학회 2000 대한임상병리학회지 Vol.20 No.1

Development of a Novel Long-Range 16S rRNA Universal Primer Set for Metagenomic Analysis of Gastrointestinal Microbiota in Newborn Infants

( Hye Jin Ku ),( Ju Hoon Lee ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.6

Metagenomic analysis of the human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of the lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new longrange 16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias.

중합효소연쇄반응과 16S rRNA 염기서열을 이용한 토착호산성박테리아의 동정

김봉주,박천영,위대웅,백근식,성치남,최낙철 한국자원공학회 2012 한국자원공학회지 Vol.49 No.4

Polymerase chain reaction and 16S rRNA sequence were investigated to identify the indigenous acidophilic bacteria from the Goseong and Yeonhwa in Korea, and the Japanese bacteria in Japan. In comparison sequences with the type strain of Acithiobacillus ferrooxidans(ATCC 23270), the similarity of Goseong and Yeonhwa bacteria were obtained the range of 96.99-97.79% and 97.59-97.90%, respectively. By comparison sequences with type strain both A. ferrooxidans and A. caldus(ATCC 51756) the similarity were founded range of 97.26-97.97% and 93.84% in the Japanese bacteria, respectively. The similarity of these bacteria were found 98.5% with the type strain of Acithiobacillus ferrooxidans by using DGGE in the cluster analysis. When these bacteria were incubated in growth-medium with and without sulfur, the pH value were rapidly decreased more containing ferrous iron medium than medium with sulfur. 성, 연화 및 일본 토착호산성박테리아를 동정하기 위하여 중합효소연쇄반응과 16S rRNA 염기서열 분석을 수행하였다. 3개 지역 시료에서 얻어진 16S rRNA 염기서열을 표준균주인 Acidithiobacillus ferrooxidans(ATCC 23270)와 pairwise 비교 정렬(alignment sequence) 방법으로 유사도를 비교하였다. 표준균주와 유사도를 비교한 결과, 고성 및 연화 박테리아의 염기서열은 유사도가 각각 96.99-97.79%와 97.59-97.90%로 나타났다. 일본 박테리아는 표준균주와의 유사도가 97.26-97.97%로 나타났고 A. caldus 표준균주와는 93.84%의 유사도를 보였다. 변성구배 겔 전기영동으로 3개 지역 박테리아들에 대한 군집분석을 수행한 결과 고성, 연화 및 일본 박테리아는 모두는 Acidithiobacillus ferrooxidans와 98.5%의 유사도를 보였다. 이들 박테리아들을 이용하여 성장-실험을 수행한 결과 원소 황을 포함하는 배양액에서 보다 ferrous iron을 포함된 배양액에서 더 빠르게 pH가 감소하였다.

Comparison of 16S rRNA Gene Sequencing, API 20E, and RT-PCR for Identification of Autopsy-derived Escherichia coli and Shigella spp

장미화,이해용,김주영,이원해,정종민,문서현 한국과학수사학회 2022 과학수사학회지 Vol.16 No.3

Practical techniques to differentiate Escherichia coli from Shigella spp. remains underexplored in the field of forensic microbiology. We aimed to compare the performance of 16S rRNA gene sequencing, and two commercially available API 20E and RT-PCR systems to distinguish between E. coli and Shigella spp. on the basis of analysis of 24 isolates collected from 12 forensic autopsies to evaluate the level of species identification. The accuracy of 16S rRNA gene sequencing at the species level was ranged from 16.7% to 33.3%. However, 79.2% (19/24) were unidentified, even though analyzed using three distinct databases (MicroSEQ 500 library, BLAST, EzTaxon). Phylogenetic analysis confirmed that 16S rRNA gene sequencing could not differentiate Shigella spp. and E. coli. Of the 19 isolates, the API 20E showed complete identification of 78.9% (15/19) at the species level, whereas RT-PCR analysis showed complete identification of 19 isolates (100%) at the species level. Our comparison analyses revealed that only 16S rRNA gene sequencing is not enough to differentiate E. coli and Shigella spp., and the accuracy and rapidity order was as follows: RT-PCR >API 20E>standard analysis. These results suggest that the polyphasic strategy with the standard analysis and RT-PCR or API 20E techniques are ideal for differentiation of these microbes.

제주마 고환내 세균의 16S rRNA 염기서열 분석을 이용한 동정

박용상,김남영,한상현,박남건,고문석,조원모,채현석,조인철,조상래,우제훈,강태영 한국임상수의학회 2014 한국임상수의학회지 Vol.31 No.1

Many bacteria colonized in the horse semen affect quality of the sperm and some may cause infectionin the mare reproductive tract and infertility of susceptible mare. This study was initiated to determine the prevalenceof bacteria in testes of Jeju horses by determining rRNA sequence. The samples were swabed from the testes of nineJeju horses (aged from 8 to 12 months after birth). Bacteria isolated from testes were identified by 16S rDNA sequencing. 1.6-kbp PCR products for 16S rRNA coding region were obtained using the universal primers. The PCR productswere further purified and sequenced. Maximum similar species were found by BLAST search in the GenBank DNAdatabase. BLAST results showed that the sequences were similar to those of Acinetobacter sp (A. schindleri, A. ursingii).,Bacillus cereus, Corynebacterium glutamicum, Escherichia coli, Gamma proteobacterium, Micrococcus luteus,Pseudomonas mendocina, Shigella sonnei, Sphingomonas sp., Staphylococcus sp (S. cohnii, S. saprophyticus, S. xylosus)., and Stenotrophomonas maltophilia. DNA sequences for 16S rRNA is provided useful informations for speciesidentification of pathogenic microorganisms for the reproductive organs in horses.