NC ｜ Wang Jun / song Mozhi combined with third-generation sequencing to analyze the structural variation and function of intestinal flora

Periodical ：Nature Communications

link ：https://doi.org/10.1038/s41467-022-30857-9

- Research background -

The continuous progress of sequencing technology is an important driving force for the development of life science , The rise of microbiome field depends on the wide application of second-generation sequencing technology . Especially the intestinal microbiome , Its role in human health and disease has been paid more and more attention , In the past decade, it has become a hot spot in the field of life science .

At present, a large amount of data have been accumulated on the composition and function of human intestinal microbiome , However, most of the current studies focus on the use of second-generation sequencing technology to analyze species and functions , The quality of metagenome splicing is not high, and it is difficult to achieve strain level functional difference analysis . The gut bacterial genome is an open genome , They can be combined 、 Transformation and transduction to obtain foreign genes for gene recombination and large structural variation （Structural variations,SV）, Cause changes in bacterial function , Thus affecting human health and disease status .

There have been a series of studies using second-generation sequencing methods to detect deletions in the intestinal flora （deletion） Dominant SV The function of the system is characterized , And prompt these SV It has an important effect on the function at the strain level ; However, the reading length of the second generation sequencing technology is limited , Such methods rely on established bacterial reference genomes , Detect insertion （insertion） Or inversion （inversion） Limited ability , And there are some difficulties in verification .

Third generation sequencing technologies such as ONT and Pacbio And the new generation sequencing technology has a longer reading length （ You can get 10kb-1Mb fragment ） The advantages of , It can help to better splice various complex genomes and metagenomes , And it plays an important role in genome optimization of many organisms , For example, the human genome T2T（telemere to telemere） etc. .

- Research progress -

For intestinal metagenome research , Wang Jun, research group of Institute of Microbiology, Chinese Academy of Sciences, and song Mozhi, research group of Institute of zoology, Chinese Academy of Sciences , Established ONT Third generation sequencing and Illumina Second generation sequencing data hybrid assembly and subsequent analysis process .

① Hybrid assembly improves assembly quality and SVs Detection capability

stay mock community The verification on shows that , The hybrid assembly of third - and second-generation sequencing data from integrity 、 In terms of accuracy and coding density, it has more advantages than the simple second-generation or third-generation sequencing assembly . The researchers did 100 Personal cross section 、10 personal 10 Sequencing analysis of samples at consecutive time points , whole contig Of N50 Compared with the second generation sequencing, it has improved 3 times ; By dividing the boxes, we get N50 by 117kb Of 9,612 individual MAGs（ Metagenomes assemble genomes ）, cover 692 Species , These include those that have not yet been assembled 69 Bacterial genomes . At higher abundance 、 Representative MAGs Through comparison , A total of 317,558 Insertional mutations ,34,129 Deletion mutations and 1,373 Gene inversion （ chart 1d）; And this SV The distribution of has taxonomic differences ,Firmicutes Doory SV most , and Desulfobacteroita Minimum door . Randomly selected SV Further through the matching verification of three generations of sequencing , Find out 97% Of SV Long sequences can be directly used to verify the existence of .

chart 1 (a) The technical route of this study ： Using three generations of sequencing SV Deep analysis of , And cross section / In time series SV The composition of 、 dynamic analysis , In the end SV Determination of the effect on metabolic function .(b, c) Hybrid assembly can effectively improve N50, And assemble a large number of genomes .(d) There are more insertion、deletion and inversion.

② SVs“ The fingerprint ” Different hosts can be distinguished

The analysis of cross section and time series also shows that ,SV It can be used as a sign to distinguish the same bacteria from different people , This is consistent with recent reports . Cross sectional study , Every... Between different individuals Mb The average genome contains 16.7 Of SVs, The same individual at different time points Mb Genome species SVs The median value of is 0, Confirmed that SV Stability in time .

③ SVs Cause changes in gene function at the strain level

To include SVs Functional enrichment analysis of related genes , stay top30 Found in the passage 19 Pathways and polysaccharide degradation 、 Sphingolipid metabolism and other metabolic functions , And grow with cells 、 There are very few replication related pathways . The study group also examined the feces of a cross-sectional cohort 、 Metabonomic analysis was performed on serum and urine samples , And through correlation analysis, we found SVs It leads to possible changes in the function of the upper generation at the strain level . for example ,Fusicatenibacter saccharivorans And fecal samples Neotrehalose Concentration has a significant correlation （ chart 3d）; But some genes have SV For example, in case of insertion or missing , Some of the strains lost significant association with metabolites . And F.saccharivorans There was also a significant negative correlation with fasting blood glucose , But with SVs In subgroup , The correlation becomes insignificant （ chart 3h）. Again ,SVs The existence of also makes A.rectalis And glucose When the correlation is significant at the overall strain level , Disappear at strain level ( chart 3i).

chart 3 Intestinal microbes are associated with SVs Relevant functional research results .(a, b, c) SV Affect gene enrichment results ; (d-i) SV Affect the relationship between different strains in a single strain, metabolites and blood glucose .

④ Community prophage And CRISPR The structure is highly correlated

Last , For possible SV Of prophage as well as CRISPR spacer Further analysis of the system , Found out 2,247 Protophage （ chart 3a） as well as 1,077 Protophage - Host correspondence （ chart 4b）; Only one 72 Included in the existing MVP In the database ; For all MAGs The analysis found that 150,058 individual CRISPR spacers, Average of each sample 1,665 ± 560 individual spacers, And only 11.73% Already in CRISPROpenDB Database summary . More interesting ,Prophage/CRISPR spacers Of β Diversity is significantly correlated , In addition, there was a significant negative correlation between the composition of protophage and virus among different individuals in the cross-sectional cohort （ chart 4c）.

chart 4 The relationship between viruses and CRISPR Relevant research results

- Research Summary -

in summary , This article is based on three generations ONT Sequence improves the quality of macro gene assembly 、SV The ability to discover , A large number of structural variations including insertion mutations and gene inversion have been found to affect gene function at the strain level , And bacteriophages 、CRISPR-spacer And so on . The research group used three generations Nanopore Sequence analysis of enterovirus group （Cao et al., Medicine in Microecology, 2020, 4:100012; Cao et al. Gut Microbes, 2021, 13）, A recently published method for the analysis of Mycota （Lu et al, Molecular Ecology, doi:10.1111/mec.16534）, And targeting RNA Detection of pathogenic microorganisms （Zhao et al., Advanced Science, 2021, 8, 2102593） after , Using three generations Nanopore Sequencing technology explores the new progress in the research field of intestinal microorganisms .

- Corresponding author -

Institute of Microbiology, Chinese Academy of Sciences

king army

Doctor of Science , Doctoral supervisor

Wang jun, , Institute of Microbiology, Chinese Academy of Sciences , Researcher of the Key Laboratory of immunology and pathogenic microorganisms , Partner group of German Max Planck Institute - Leader of bioinformatics and computational biology , Overseas high-level talent support program youth program candidates .

He has long been engaged in bioinformatics and computational biology research with microbiome as the core , In recent years, a series of important advances have been made in the research and development of detection and treatment methods based on symbiotic microorganisms in important infections and chronic diseases , It mainly includes ：

• Combining new sequencing methods and new artificial intelligence analysis techniques , Auxiliary pathogen detection and infection treatment .

• For major infectious diseases and immune diseases , In depth analysis of intestinal microbiome characteristics and correlation .

• utilize GWAS Research identifies key interactions with microorganisms 、 Host genes that maintain intestinal microflora homeostasis .

As corresponding author or first author in Nature Biotechnology、Science、Nature Genetics（2 piece ）、Cell Host & Microbe、Advanced Science、Nature Communications（2 piece ）、Microbiome（2 piece ）、Protein & Cell（3 piece ）、Arthritis & Rheumatology、Gut Microbes And so on 60 Yu Wen （ Including published in Science、Nature Genetics Inside 6 Cover article ）, Total impact factor >1000, quote 8400 Remaining times （Google Scholar data ）; Apply for domestic patent 5 term （ to grant authorization 1 term ）,PCT patent 1 term . Undertake and complete the major research plan cultivation project of NSFC 、 Special project of the NSFC for emergency research on novel coronavirus , Served as the project leader of the national key R & D plan , Project leader of key deployment projects of Chinese Academy of Sciences ;2020 year 3-4 In May, he participated in the anti epidemic research on behalf of the Institute of Microbiology in Wuhan .

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