Microbial Community Analysis II

Question 1

What is meant by metagenomics?

Answer 1

The study of genetic material recovered directly from environmental samples

Question 2

What technologies can be used to analyse whole microbial communities (culture-independent)?

Answer 2

Pyrosequencing (454)
MiSeq
HiSeq

Question 3

What does the random shotgun approach provide information on?

Answer 3

Provides information on the whole genetic potential (function) and identity (phylogeny) in an environmental sample

Question 4

What is the molecular target of metagenomics?

Answer 4

Chromosomal genomic DNA

Question 5

What knowledge is gained from metagenomics?

Answer 5

High resolution microbial profiling

Gene contents from uncultivated microbes

Question 6

What are the limitations of metagenomics?

Answer 6

No information on microbial expressed functions

Question 7

What are two commonly used pyrosequencing systems?

Answer 7

GS FLX and GS Junior Systems

Question 8

What starting materials can be used in these systems?

Answer 8

Genomic DNA, PCR products, BACs and cDNA

Question 9

How can a sample be prepared for 454 or MiSeq experiment?

Answer 9

Begin with a set of barcoded primers e.g. 8 forward primers, 12 reverse primers (each containing different barcode), to create 96 combinations = 96 individual samples or libraries
For each each environmental DNA sample, use a unique forward and reverse primer pair to perform PCR
End up with a PCR product (amplicon) of the 16S rRNA gene (portion of it as dictated by the primer pair) of all the bacteria comprising your environmental sample
Pool all the PCR amplicons (for each environment sample) and send to a 454 or MiSeq sequencing facility

Question 10

How does the 454 Genome Sequencer FLX system work?

Answer 10

Each bead contains a cDNA short sequence to bind the adapter
The system works in a way that allows only one PCR amplicon (from the pooled environmental samples) to bind to just one bead
An emulsion is created whereby PCR takes place to amplify each DNA sequence, and as the sequence on each bead is amplified, the newly formed sequences attach to the bead
Essentially you end up with millions of copies of a single clonal fragment contained on each DNA Capture Bead
Each bead then gets loaded into one individual well on a 454 Titanium plate i.e. one bead per well
The chemistry of 454 is such that the DNA strands on each bead get sequenced - essentially, each well is a DNA sequencing machine
There are 1 million wells on the plate, which means you theoretically end up with 1 million sequenced strands
PTP device loaded in instrument for sequencing
Individual nucleotides are flowed in sequence across the wells
Each incorporation of a nucleotide complementary to the the template strand results in a chemiluminescent light signal recorded to the camera

Question 11

Compare 454 and MiSeq

Answer 11

454: 
~1 million sequence read output
~450bp read lengths
Cost ~£10,000
MiSeq:
~15 million sequence read output
2 x 300bp read lengths
Cost

Question 12

What is the outline of the de-multiplexing process?

Answer 12

Extract DNA and PCR amplify with barcoded primer (pool amplicons) > Pyrosequence amplicons using 454’s GS FLX instrument (screen, assign sequences to samples using barcodes) > Align to reference alignment (e.g. using NAST), infer phylogeny (e.g. using FastTree) > tens of thousonds of sequences, 100s of samples (fast UniFrac) > Cluster samples based on UniFrac distances

Question 13

What are OTUs?

Answer 13

Operational taxonomic unit. Species distinction in microbiology, typically using rRNA and a percent similarity thereshold for classifying microbes within the same, or different, OTUs
Allows determination of species richness of an environmental sample

Question 14

How does Illumina/MiSeq work?

Answer 14

Done by a process called “sequencing by synthesis”

• Input DNA is tagged with adaptors during “tagmentation” and are denatured to make them single-stranded
• DNA fragments are washed across a flow slide
• DNA hybridises to complementary oligos on the surface of flow-slide and DNA that doesn’t attach is washed away
• DNA attached to flow-slide is replicated to form small clusters of DNA with the same sequence
• unlabelled nucleotides and DNA polymerase are added to lengthen and join the strands of DNA attached to the flow slide, creating “bridges” of dsDNA between oligos and flow slide surface
• dsDNA denatured to ssDNA leaving several million dense clusters of identical DNA sequences
• a fluorescently-labelled reverse terminator is imaged as each dNTP is added and then cleaved to allow incorporation of the next base
• since all 4 reversible terminator bound dNTPs are present during each sequencing cycle, natural competition minimises incorporation biased