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Metagenomic course - Experimental design and sequencing technologies
Transcript of Metagenomic course - Experimental design and sequencing technologies
Next Generation Sequencing
Metagenomics: experimental design and sequencing technologies
Start of Human
Max read length PE 300 cycles
Lower cost per base
Error rate ~1%: only substitutions, no indels
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)
31th March 2014
Roche - 454
Illumina - Genome Analyzer
Illumina - HiSeq
ABI - SOLiD
(sequencing by ligation)
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
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
Temperton & Giovannoni (2012)
10^30 microbial cells on Earth
Most don't grow in the lab
Search for genes and functions
Samples must be
How many & which samples?
Q: scale and amplitude of the variation?
Key design decisions
Special samples and treatments:
is comparison with other samples possible?
Knight et al (2012) Nature Biotech.
- Lab culture
- PCR amplicon + Sanger seq
- PCR amplicon + NGS (16S, ITS, other genes)
- NGS shotgun 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.
Illumina 16S sequencing
Illumina human saliva 16S dataset
BWA-mem – Silva
BWA-mem – GreenGenes
CLC – GreenGenes
How critical is DNA isolation?
can be introduced:
prep protocol, primers, PCR cycles, polymerase...