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Metagenomic course - Experimental design and sequencing technologies

Nijmegen (NL) 31st March 2014
by

Adalberto Costessi

on 29 January 2015

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Transcript of Metagenomic course - Experimental design and sequencing technologies

Manager
Next Generation Sequencing
ADALBERTO
COSTESSI
Metagenomics: experimental design and sequencing technologies
Start of Human
Genome Project
Metagenome technologies
Sample multiplexing
Max read length PE 300 cycles
Lower cost per base
Error rate ~1%: only substitutions, no indels
Experimental design
2
1
Adalberto Costessi
Manager NGS
BaseClear

adalberto.costessi@baseclear.com
www.linkedin.com/in/costessi
Questions ?
Background:

2003: MSc Medical Biotechnology, University Trieste (Italy)

2004-2005: European Space Agency (NL)

2010: PhD Molecular Biology, Henk Stunnenberg lab, Radboud University Nijmegen

Last 3 years: Manager NGS, BaseClear BV, Leiden (NL)
http://www.nature.com/nature/journal/v458/n7239/full/nature07943.html
http://www.genome.gov/sequencingcosts/
31th March 2014
H. influenzae
first sequenced
free-living organism
http://www.wellcome.ac.uk/Education-resources/Education-and-learning/animations/dna/wtdv026689.htm
Roche - 454
(pyrosequencing)
Illumina - Genome Analyzer
(sequencing-by-synthesis)
Illumina - HiSeq
(sequencing-by-synthesis)
ABI - SOLiD
(sequencing by ligation)
First draft
human genome
founded
Independent company founded in 1993
Based in Leiden, NL
ISO17025 & GMP analyses
Service & solution provider:
Sanger sequencing and microbial IDs
Forensic & genotyping
Genome analysis: Next-generation sequencing & Synthethic biology

PacBio RSII

true single molecule sequencing
sequences up to 15 kb

genome sequencing and scaffolding
Iso-seq: complete transcripts and splice variants

Illumina HiSeq2500 & MiSeq

massively parallel sequencing (100s mlns reads)
short reads (50-300 nt)

genome sequencing and analysis
transcriptomics
metagenomics
Temperton & Giovannoni (2012)
Why metagenomic?
10^30 microbial cells on Earth
Most don't grow in the lab
Search for genes and functions
Quality control
Forensic

Samples must be
representative
How many & which samples?
Q: scale and amplitude of the variation?
Key design decisions
=
Biological/technical replicates?
Interpretation?
Informative?
Deep enough?
Special samples and treatments:
new protocols?
new biases?
is comparison with other samples possible?
vs.
Knight et al (2012) Nature Biotech.
http://www.nap.edu/openbook.php?record_id=11902&page=12
Approaches:

- Lab culture
- PCR amplicon + Sanger seq
- PCR amplicon + NGS (16S, ITS, other genes)
- NGS shotgun sequencing
PacBio sequencing
True single molecule w/o PCR amplification
Very long reads:
median of 4-5 kb, up to 15kb
High error rate ~13-15%, but unbiased
8 SMRT cells per run
~10-50k reads/SMRT cell
Multiplexing is still challenging
Input DNA: 1-5 ug
1 SMRT cell = thousands of zero-mode waveguides (ZMWs), of tens of nanometers in diameter.
In each ZMW a single DNA molecule is detected.
PacBio
Illumina 16S sequencing
DNA isolation
Illumina human saliva 16S dataset
BWA-mem – Silva
BWA-mem – GreenGenes
CLC – GreenGenes
How critical is DNA isolation?
Sample 1
Sample 2
Kit 1
Kit 2
Kit 3
Kit 4
Recent project
PacBio RSII
(sequencing-by-synthesis)
Sampling challenge
Bias
can be introduced:
prep protocol, primers, PCR cycles, polymerase...
BIAS BOX
Full transcript