Metagenomic

Shotgun Sequencing
Learning Progress
 1. Metagenomic approach  A non-culture
based approach to analyse microbial
communities
2. Shotgun Sequencing 
unrestricted/untargeted genome sequencing

What it is?
 Why it is needed?

Obtain information on:

1. Microbial species
2. Microbial
functions/pathways
Who? & What? & How ? 3. Putative genes
1. FMT
1. Clinical 2. Personalized
interpretation nutrition
2. Biomarker discovery 3. Modulation of
3. Phylogeny gut microbes
reconstruction profile  diet &
lifestyle
modification
Advantages over 16S rRNA Sequencing
• Enhanced detection of bacterial species
• Increased detection of diversity (Shannon diversity, Simpson Index,
Evenness)
• Increased prediction of genes

Disadvantages
- Greater risk of long-range missassembly
How it is?
What it could serve?

Sharpton et al. (2014)

About the project
Aim(s)
• to obtain the difference of the taxonomic relative abundance of the
gut microbiota between different strategies in NGS, which are 16S
rRNA and Shotgun sequencing
Research Questions?
• How deep the metagenomic shotgun sequencing provides the gut
microbiota relative abundance data regarding the level of taxonomy
compared to 16S rRNA sequencing?
• Which strategy that has more advantages or disadvantages in terms
of describing the taxonomic relative abundance of the microbiome?
• Are there any missing spaces in one strategy that could be filled by
the other strategy regarding the relative abundance of the gut
microbiota?
Study design

Amplicon-
16S rRNA Sequencing based
Analysis
Extracted DNA
from Malinau Taxonomic Relative abundance
Compared
Sample (n=4) Analysis of Diversity

Metagenomic shotgun Contig-based

Sequencing Analysis
Reference
Pipeline for
metagenomic
analysis

Sharpton et al, 2014

 Another pipeline reference
Contig
Reads Filtration Assembling
Filtered contig Assembled contig
(FastQ) (FastX tool) (MetaVelvet
Assembler)
Reads < 75 nt,
Phred score < 20
Reads < 90% identity & e-value 1e^-3
Queried against
Reads align to human genome
NCBI-nt database

90% identity & e-value 1e^-3

BLAST outputs
Relative abundance
taxa

Ranjan et al, 2016

Proposed Workflow
 DNA Fragment
DNA Extraction DNA Purification DNA Shearing
Analysis
QIAmp Power Fecal DNA QIAmp Power Fecal DNA DNA Shearing for Next- 1. Agilent DNA 1000 kit on the 2100
Isolation Kit Isolation Kit Generation Seq. with Bioruptor Bioanalyzer instrument
pico, 0.2 ml microtubes (Protocol 3)
(Protocol 1) (Protocol 1)
(Protocol2) 2. Agarose Electrophoresis
(Protocol 4)
3. Qubit dsDNA BR Assay Kit
(Protocol 5)
Repairing ends and
Validate Libraries Ligate Adapters Adenylate 3’ ends
Library size selection
Qubit dsDNA HS Assay Kit TruSeq DNA PCR-Free HT TruSeq DNA PCR-Free HT TruSeq DNA PCR-Free HT
(Protocol 9) Library Prep Kit Library Prep Kit Library Prep Kit
(Protocol 8) (Protocol 7) (Protocol 6)

Normalize and Pool Denature and Dilute Load Libraries onto

Run Sequencing
Libraries Libraries Reagent Cartridge
TruSeq DNA PCR-Free HT Illumina NextSeq Hi Illumina NextSeq Hi
Library Prep Kit Output KT V2 (300cycle) Output KT V2 (300cycle)
(Protocol 10) System System
(Protocol 11) (Protocol 12)

Metagenomic Data set

(FastQ)
Wet lab work
 Metagenomic Data set
(FastQ)

Metagenome
Adapters removal Data trimming Contigs binning
assembling
Quality Control 1. De Bruijn graph approach 1. CONCOCT
2. Meta-IDBA 2. GroopM (Imelfort et al.
1. Cutadapt (Martin) 2014)
3. MetaSPAdes Assembler
2. Sickle (Joshi and Fass, 2011) 4. 3. MetaBAT
Ray (Distributed assembler) 4. Combined approach
3. FastqMcf (Aronesty, 2011) 5. MEGAHIT (1/2/3+Anvio)

Composition Contig-based Metagenome

classification profilling profilling
???? 1. CLC genomic workbench 1. Kraken
(de novo assembly algorithm) 2. Web-based
(Ranjan et al, 2016) + combine metagenomic
analysis provider :
Relative abundance Assembly-based MG-RAST, LEfSE,
taxa profilling MetaPhIAn, NBC,
etc.

Marker gene
Dry lab work Analysis
Next week…
1. Understanding the wet lab protocols
2. Getting familiar with bioinformatic tools
3. Completing the draft of literature review & methods