Microbial Genomics

Genome sequencing has come of age, and

genomics will become central to microbiology‘s
future.

Carl Woese
(the microbiologist who defines the kingdom Archae)

-What is microbial genomics ?

Microbial genomics is an important field by utilizing nucleic acid sequencing
technologies to investigate the genomic information of a single microbial species
or microbial communities from a wide spectrum of environments.
-The application of microbial sequencing
identifying drug target, farm animal health,
overcoming antibiotic 输入 aquaculture,
resistance and some vegetables and fruits
diseases, etc. production, etc.

输入 输入

food industries novel energy

(such as fermented production
foods and liquors 输入 systems,
industries), some pollution
emerging industries, control, etc.
etc.
 01
Backgrounds

Content 02
Applications and classifications

03
Prospect of microbial genomics
01 Backgrounds - Microbes

A microorganism or microbe, is a
microscopic organism, which may
exist in its single-celled form, or in a
colony of cells. They are about one
tenth the size of a typical human cell.
They are found all around us, and
even inside our bodies.

The category “microbes” includes

prokaryotic microbes (bacteria and
archaea), eukaryotic microbes (fungi,
protozoan, and algae), and acellular
microbes (virus, virusoid, viroid, and
prion).
The magnitude of microbial diversity
 The weapon: next-generation sequencing
PCR Read TH/run;
Platform Chemistry Reads/run Disadvantages Applications
amplification length Run time
Sequencing-by- EmRCR >400 1,000,000 0.4- PCR biases; asynchronous De novo genome
synthesis 0.6Gb; 7- synthesis; homopolymer sequencing, RNA-seq,
Roche (Pyrosequencing) 10h run; base insertion and resequencing/targeted
454 deletion errors; emPCR IS resequencing
cumbersome and
technically challenging
Polymerase- Bridge 75/2 × 40,000,000 3-6/200* PCR biases; low De novo genome
based amplification 100a Gb; 3-4 multiplexing capability of sequencing, RNA-seq,
Illumina sequencing-by- days samples resequencing/targeted
synthesis resequencing,
metagenomics, ChIP
Ligation-based Em PCR 35-40 85,000,000 10-20Gb; EmPCR is cumbersome and Transcript counting,
SOLiD sequencing 7 days technically challenging PCR mutation detection, ChIP,
biases; long run time RNA-seq, etc.

After the first commercial next-generation Sequencing (NGS) platform Roche 454 was promoted in 2005, NGS has
revolutionized microbial taxonomy and classification and has altered the landscape of microbial genome projects. It
produces sequence data around one hundred times faster and cheaper than Sanger approach.
 The weapon: third generation sequencing

The competitive third generation technologies are already in production, which are characterized by portability (MinION), speed,
longer reads, and the allowance for direct detection of epigenetic markers. The representatives of the third generation are
PacBio‘s SMRT and Oxford Nanopore’s MinION. GenoCare prototype was developed by DirectGenomics as the first clinical third
generation sequencing platform in 2015.
• Although the third generation sequencing technology is largely limited by relatively high error rate and high cost,
the speed of sequencing is important and promising in the clinical setting to allow for timely diagnosis and clinical
actions.
• The relatively long reads permit a near-complete viral genome sequencing directly from a primary clinical sample
with high accuracy (about 97-99% identity).
• In addition, the third generation sequencing technologies (including both MinION and SMRT platform) have been
used in the 16S ribosomal RNA gene sequencing, and it turns out that the error rate of PacBio is comparable to
that from the next generation sequencing platforms, such as 454 and Illumina MiSeq.
 The Applications of Sequence Data
01 De novo assembly of entire genomes to generate primary genetic
sequences for the detailed genetic analysis of an microbial organism.
02 Resequencing for the discovery of variants that differ to known genome
sequences of a closely related strain.
03 Species classification and novel gene discovery in genomic surveys of
microbial communities.
04 “Seq-based” assays that determine the genomic content and abundance
of mRNAs, small RNAs, and non-coding RNAs (RNA-seq) or measure
profiles of DNA-protein complexes, methylation sites, and DNase I hyper-
sensitity sites.
02 Applications and Classifications
 Whole genome sequencing

Whole genome sequencing here refers to de novo sequencing of microbial genomes. The availability of complete genome
sequences of closely related organisms provides a possibility for reconstructing events of genome evolution.
Roche 454 has been generally considered more fit for genomes containing abundant repeated sequences due to the
production of larger fragments over 400 bp. However, Illumina/Solexa platform has been improved for eukaryotic de novo
sequencing by (i) the longer read length using better extension reagents and chemistries; (ii) the development of paird-end
tag sequencing, in which both ends of fragments are sequenced to offer more information; and (iii) novel assembly
algorithms that deal with numerous short reads.
Genotype-phenotype association mapping
 RNA-seq has been a central approach
to profiling mRNA populations. The
traditional techniques, such as
microarrays, have several limitations.
First, they can not detect low-
abundance transcripts. Second, the
discovery of novel transcripts is
limited. However, NGS platforms-
RNA-seq based RNA-seq can get over these
drawbacks. This methodology allows
us to annotate transcripts, including
protein encoding sequences and
non-coding sequences; determine
the transcriptional structure of genes;
and quantify the expression level of
transcripts under specific conditions.
ChIP-seq

Chromatin immunoprecipitation
sequencing can provide whole-
genome mapping of DNA-binding
protein sites. This approach has
become an indispensable tool for
investigating gene regulation and
epigenetic mechanisms.
 Metagenomics
Metagenomics, also known as community genomics,
provides the solution for cultivation bottleneck, by
assessing the genetic content of an uncultured
microbes. The brief process includes the isolation of
DNA from environmental samples, the cloning of
DNA into artificial bacterial chromosome, Plasmid, or
Fosmid, and the following sequencing.

The great plate count anomaly. The inability to isolate and

cultivate many types of microorganisms has long limited the
microbial studies. The cultivable fraction of microbes is low, less
than 1%-a remarkable phenomenon known as “The Great Plate
Count Anomaly”.
The construction of a metagenomic library.
Applications of metagenomics

1. Determination of microbial diversity

2. Discovery of novel pathways
3. Exploration of the diversity of targeted genes
4. Identification of traits of uncultured microbes
5. Patterns of community versus population diversity
The challenges of metagenomics

1. The complexity of sequencing data

2. The recovery of sufficiently purified high-molecular-weight DNA
without bias.
3. The unequal abundance of community members.
4. The assembly of genomes
 03 Prospect of microbial genomics
i. Strategies
Although powerful, metagenomics is limited by
1 fragmented nature of metagenome assemblies for
short reads and by the lack of single-cell resolutions.
While metagenome assemblies often collapse due
Prospect 2 to strain heterogeneity, single-cell genomics has the
ability to get it over.
Single-cell sequencing and analysis workflow for
3
standard (untargeted) and targeted single-cell
sequencing strategies. Targeted strategies illustrate
both phylogeny-driven and function-driven
methods.
ii. Computational methods

Over the last decade, there has been more than a 90% reduction in the cost of genomics technologies.
Faced with much more data than we can analyze, computational methods for comparative analysis
must be implemented. The most promising approach for this bottleneck involves a conceptual change,
namely, the realization that effective comparative analysis do not require to compare all genes with all
other genes.

iii. Future directions

Since the first complete microbial genome (Haemophilius influenzae) was published in 1995, a
dramatic rise in the number of sequenced microbial species has occurred. Genomics focuses are
gradually moving to proteome and metabolome, in effort to explore cellular interactions, ecology and
evolution.
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