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Application of high-throughput sequencing technology in the study of intestinal flora diversity
There are trillions of commensal flora in the human intestines. They come in a variety of species, up to thousands, and the number is amazing. It is more than 10 times the total amount of human cells. So far, there are still more than 80%. Microorganisms are not known. These gut microbes and human beings have a mutually beneficial relationship and play an important role in maintaining human health. They maintain a dynamic balance in the intestines, which can synthesize vitamins, help the body absorb nutrients from food, maintain intestinal immune system function, and resist harmful microbes, but when this balance is broken due to certain factors, the intestines When the flora is disordered, the human body may develop diseases such as obesity, diabetes, enteritis and even cancer. In order to deepen the understanding of the causes of human diseases and the mechanism of action of drugs, following the Human Genome Project, scientists have launched the Human Genome Project, which has ushered in a new wave of research on intestinal flora.
In the past studies on the diversity of intestinal microflora, people mainly used molecular biology methods such as DGGE and biochips to study the intestinal flora, but these methods have certain defects. For example, DGGE can only detect more than ten dominant bacteria in environmental samples, but it is helpless for trace microorganisms; the electrophoresis band contains more than one 16S rDNA sequence, and it is necessary to clone and sequence the specific species information. The experimental operation is cumbersome; in addition, this method does not reflect the abundance of microorganisms. The biochip obtains microbial diversity information through probes fixed on the chip. "It can only verify the known, but can not explore the unknown." It is not very accurate to judge the abundance of microorganisms by the strength of the signal. Since its inception in 2005, a new generation of high-throughput sequencing technology has been widely used in the study of intestinal flora with its digital signal, high data throughput, high sequencing depth and high accuracy. More than 60 articles, including many articles published in top international journals such as Nature, PNAS, Genome Res, Gut, Gastroenterology. The Roche 454 sequencing platform is the best platform for microbial diversity research because it has a long read length of 300-500 bp and can span one or several variable regions of 16S rDNA sequence. The paper published on the platform for intestinal flora More than 50 articles. Mehki Biotech has a Roche 454 sequencing platform and has accumulated a large number of successful cases in the study of intestinal microbial community diversity.
Through reading a large number of related literatures, Meiji Biologists have found that the application of high-throughput sequencing technology in the study of intestinal flora diversity focuses on the following aspects: 1 diet, energy intake, obesity and intestinal flora Relationship between; intestinal flora and disease occurrence; 3 effects of antibiotic treatment on intestinal flora; 4 intestinal microflora structure of different individuals and other external factors (pressure, pathogenic infection, The impact of surgery). Some of these articles are described below:
First, the relationship between diet, energy intake, obesity and intestinal flora
1. A core gut microbiome in obese and lean twins - 2009 "Nature"
Turnbaugh PJ et al. studied intestinal microbes in 154 individuals with different fat and thin identical twins (31 pairs), fraternal twins (23 pairs) and their mothers (46). The V2 and V6 regions of the microbial 16S rRNA gene in these individuals were determined using a 454 high-throughput sequencing platform, and 18 individuals were selected to determine the DNA of the intestinal microflora. The results showed that obesity was associated with changes in gut-level microbes. Compared with lean people, microbial diversity was significantly reduced, Bacteroidetes accounted for a lower proportion and actinobacteria accounted for a higher proportion. . Shotgun reads are compared with multiple data and found that different individuals contain a large number of shared microbial genes, which can be defined as "core microbiome" at the genetic level, and the deviation of microbes from the core microbiome in the individual is A variety of different physiological states (such as fat and thin).
2. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa - 2010 "PNAS"
To study the effects of diet on gut microbiota diversity, Filippo CD et al. used 454 high-throughput sequencing technology to compare the composition of gut microflora in 15 healthy European children (EU) and 14 healthy African rural children (BF). . The EU diet has typical Western characteristics, while the BF food represents the traditional African countryside diet. The researchers amplified 29 S rRNA gene V5-V6 DNA fragments from 29 fecal samples by PCR and performed high-throughput sequencing, yielding 438,219 high-quality reads. The RDP database alignment showed that 94.2% of reads belong to Actinobacteria , Bacteroidetes , Firmicutes and Proteobacteria , but their proportion in EU and BF There are significant differences. Bacteroides in the BF gut accounted for the majority, while the proportion of wall thick bacteria was lower. Interestingly, Prevotella and Xylanibacter strains containing large amounts of cellulose and xylan hydrolyzed genes are only present in BF and have a higher proportion.
Second, the association between intestinal flora and disease occurrence
1. Gut Microbiota in Human Adults with Type 2 Diabetes Differs from Non-Diabetic Adults - 2010 "PLoS One"
In the past, there was a correlation between intestinal flora and human metabolic diseases. In order to study the relationship between diabetes and intestinal microbes, Nadja Larsen et al. studied 18 male patients with type II diabetes and 18 normal males. The V4 region of the microbial 16S rRNA gene in the feces was sequenced using 454 high-throughput sequencing technology. Sequence analysis revealed that these sequences belong to five gates of microorganisms, including Firmicutes , Bacteroidetes , phyla Proteobacteria , phyla Actinobacteria , and sputum microbes. The phyla Verrumicrobia , in which the proportion of the wall thick fungus and the Bacteroides gate is as high as 90% or more. Intestinal flora of diabetic patients in the content of the wall thickness of the door and Clostridia bacteria (Clostridia) is significantly lower than normal. In addition, the proportion of Bacteroides / Wacoides and Bacteroidetes - Prevotella /C. coccoides -E. rectale was positively correlated with blood glucose concentration, but not with body index. Betaproteobacteria is also highly enriched in diabetic patients, and such bacteria are also positively correlated with blood glucose concentrations. These results reveal that the etiology of human type 2 diabetes is associated with changes in intestinal microflora composition.
2. Towards the human colorectal cancer microbiome - 2011 "PLoS One"
Marchesi JR et al. used 454 high-throughput sequencing technology to compare the differences in microbial composition between colon tumors and normal colonic mucosa. The sequencing samples were obtained from 6 early colon cancer patients who underwent resection. The selected sequencing region was the V1-V3 region of the 16S rRNA gene. The obtained sequencing sequences were compared with the Sliva and RPD databases, and the microbial colonization patterns at two different sites were found to be surprisingly different. Although there are some differences between individual patients with colon cancer, bowel cancer tissue is always formed as a micro-ecological environment and some bacteria Coriobacteria, potentially pathogenic Enterobacteriaceae (Enterobacteria) is not representative at these sites. Studies have also found that the physiological and metabolic changes associated with colon cancer attract commensal bacteria that feed on tumors. They have a strong competitive advantage and may replace pathogens and involve the cause of colon cancer.
Third, the impact of antibiotic treatment on intestinal flora
1. Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans - 2010 "J Clin Invest"
Hematological diseases caused by highly resistant bacterial infections such as vancomycin-resistant enterococci (VRE) are an increasingly serious clinical problem, and accurate diagnosis of high-risk patients with bacterial sepsis remains challenging. Recent studies have shown that antibiotics can alter the diversity of gut microbes. In order to analyze the effects of antibiotics, Carles Ubeda et al. used VRE-infected mice as the research material and sequenced the V2 region of the 16S rRNA gene of the ileal samples using 454 high-throughput sequencing technology. The results of the study showed that antibiotic treatment of mice caused the exogenous VRE to almost completely replace the normal flora of the small and large intestine. Clinically, the researchers also found that patients receiving hematopoietic stem cell transplantation before treatment with blood, antibiotic treatment can cause VRE to "master" the intestinal flora. Antibiotics can disrupt the normal commensal gut flora and provide an opportunity for hospital infectious bacteria to "dominate" the gut flora. Therefore, analysis of intestinal microbial diversity by high-throughput DNA sequencing provides an effective means for diagnosing whether high-risk patients have bacterial sepsis.
2. The Pervasive Effects of an Antibiotic on the Human Gut Microbiota, as Revealed by Deep 16S rRNA Sequencing - 2008 "PLoS Biology"
Researchers from Stanford University used 454 high-throughput sequencing technology to explore the effects of antibiotics on human intestinal microbial community composition. They measured the sequences of the V6 and V3 regions of the intestinal microbial 16S rRNA gene of three individuals before and after taking ciprofloxacin. The obtained sequences were compared with the Silva and RPD data, and a total of 3,300 to 5,700 taxa were identified. Studies have found that taking antibiotics with ciprofloxacin reduces the richness, diversity and uniformity of human intestinal microbes, and the affected bacterial population is about one-third, but there are significant individual differences in the extent of this effect. In addition, the researchers also pointed out that after 4 weeks, the intestinal flora has basically returned to the state before the use of drugs, but some bacteria have not recovered after 6 months.
4. Intestinal microbial community structure of different individuals and their influence by external factors (pressure, pathogenic infection, surgery)
1. Composition, variability, and temporal stability of the intestinal microbiota of the elderly - 2011 "PNAS"
The human intestinal microflora structure has significant differences among the three groups of infants, adults and the elderly, which indicates that the intestinal flora has undergone tremendous changes with age. Claesson MJ et al. collected samples of 161 gut microbiota of the elderly over 65 years old and 9 adults. The V4 region of the 16S rRNA gene was determined by 454 pyrosequencing, and each individual produced more than 40,000 reads. The study found that the most dominant flora in 68% of the individual's gut is Bacteroidetes , with an average ratio of about 57%, and the proportion of Firmicutes is about 40%. However, the proportion of bacteria associated with some diseases or health varies greatly among different individuals, including Proteobacteria , Actinobacteria and Faecalibacteria . There is also a sharp difference between the core microbiome and the young people in the elderly. The former has a larger proportion of Bacteroides, and the Clostridium has different abundance patterns between the two. Analysis of 26 groups of time-0 and time-3 month stool samples revealed that 85% of the individuals had very similar microbial compositions at these two times, indicating that the gut flora of the elderly exhibited temporal stability.
2. Stressor exposure disrupts commensal microbial populations in the intestines and leads to increased colonization by Citrobacter rodentium - 2010 "IAI"
Bailey et al. used mice as a research object to explore the effects of stress on intestinal microbial community diversity and susceptibility to intestinal pathogens. The V4 region of the 16S rRNA gene of mouse intestinal microbe was determined by 454 high-throughput sequencing technology. The data obtained were compared with RPD. It was found that long-term under pressure caused an increase in intestinal anaerobic microorganisms. At the same time, the microbial richness and diversity are reduced. Among them, the relative abundance of bacteria in the genus Porphyromonadaceae , especially the genus Tannerella , is reduced. Mice were infected with Citrobacter rodentium , and the results showed that mice under long-term stress were more likely to colonize Citrobacter bismuth than normal-treated mice.
A list of articles related to the application of high-throughput sequencing technology in the study of intestinal flora diversity is as follows:
Note: 1. The article is classified from four aspects;
2. According to the influence factor of 2010;
3. The following articles all use the Roche 454 sequencing platform.
First, the relationship between diet, energy intake, obesity and intestinal flora:
1. A core gut microbiome in obese and lean twins - 2009 "Nature" - if 36.10
2. An obesity-associated gut microbiome with increased capacity for energy harvest
- 2006 "Nature" - if 36.10
3. Functional metagenomics to mine the human gut microbiome for dietary fiber catabolic enzymes - 2010 "Genome Res" - if 13.58
4. High-fat diet determines the composition of the murine gut microbiome independently of obesity - 2009 "Gastroenterology" - if 12.03
5. Composition and energy harvesting capacity of the gut microbiota: relationship to diet, obesity and time in mouse models - 2010 "Gut" - if 10.61
6. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa - 2010 "PNAS" - if 9.770
7. Human gut microbiota in obesity and after gastric bypass - 2009 "PNAS" - if 9.770
8. Interactions between gut microbiota, host genetics and diet relevant to development of metabolic syndromes in mice - 2010 "ISME J" - if 6.152
9. Use of pyrosequencing and DNA barcodes to monitor variations in Firmicutes and Bacteroidetes communities in the gut microbiota of obese humans - 2008 "BMC Genomics" - if 4.205
10. Responses of gut microbiota to diet composition and weight loss in lean and obese mice - 2011 "Obesity" - if 3.530
11. The effect of diet on the human gut microbiome: A metagenomic analysis in humanized gnotobiotic mice - 2009 "Sci Transl Med" - if 3.510
12. Energy-balance studies reveal associations between gut microbes, caloric load, and nutrient absorption in humans - 2011 "Am J Clinc Nutr"
Second, the association between intestinal flora and disease occurrence
1. Microbes and Health Sackler Colloquium: Human oral, gut, and plaque microbiota in patients with atherosclerosis - 2010 "PNAS" - if 9.770
2. The macaque gut microbiome in health, lentiviral infection, and chronic enterocolitis
- 2008 "PLoS Pathog" - if 9.079
3. Culture-independent identification of gut bacteria correlated with the onset of diabetes in a rat model - 2009 "ISME J" - if 6.152
4. Redefining the role of intestinal microbes in the pathogenesis of necrotizing enterocolitis - 2010 Pediatrics - if 5.390
5. Towards the human colorectal cancer microbiome - 2011 "PLoS One" - if 4.410
6. Gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults - 2010 "PLoS One" - if 4.410
7. Structural shifts of gut microbiota as surrogate endpoints for monitoring host health changes induced by carcinogen exposure - 2010 "FEMS Microbiol Ecol" - if 3.456
Third, the impact of antibiotic treatment on intestinal flora
1. Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans - 2010 "J Clin Invest" - if 14.15
2. Reshaping the gut microbiome with bacterial transplantation and antibiotic intake - 2010 "Genome Res" - if 13.58
3. The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing - 2008 "PLoS Biol" - if 12.46
4. Short-term antibiotic treatment has differing long-term impacts on the human throat and gut microbiome - 2010 "PLoS One" - if 4.410
5. Reproducible community dynamics of the gastrointestinal microbiota following antibiotic perturbation - 2009 "IAI" - if 4.098
6. Metagenomic analyses reveal antibiotic-induced temporal and spatial changes in intestinal microbiota with associated alterations in immune cell homeostasis - 2010 "Mucosal Immunol" - if 3.627
4. Intestinal microbial community structure of different individuals and its influence by other external factors (pressure, pathogenic infection, surgery)
1. Association between composition of the human gastrointestinal microbiome and development of fatty liver with choline deficiency - 2010 "Gastroenterology" - if 12.03
2. Depletion of luminal iron alters the gut microbiota and prevents Crohn's disease-like ileitis - 2010 "Gut" - if 10.61
3. Composition, variability, and temporal stability of the intestinal microbiota of the elderly - 2011 "PNAS" - if 9.770
4. Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors - 2010 "PNAS" - if 9.770
5. Organismal, genetic, and transcriptional variation in the deeply sequenced gut microbiomes of identical twins - 2010 "PNAS" - if 9.770
6. Characterizing a model human gut microbiota composed of members of its two dominant bacterial phyla - 2009 "PNAS" - if 9.770
7. Comparative analysis of human gut microbiota by barcoded pyrosequencing - 2008 "PLoS One" - if 4.410
8. Community-wide response of gut microbiota to enteropathogenic Citrobacter rodentium infection revealed by deep sequencing - 2009 "IAI" - if 4.098
9. Stressor exposure disrupts commensal microbial populations in the intestines and leads to increased colonization by Citrobacter rodentium - 2010 "IAI" - if 4.098
10. Intestinal microbial ecology in premature infants assessed with non-culture-based techniques - 2010 "J Pediatr" - if 4.041
11. Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags——2010 "BMC Microbiol" - if 2.960
12. Quantitative assessment of the human gut microbiome using multitag pyrosequencing - 2010 Chem Biodivers - if 1.585