Loading presentation...

Present Remotely

Send the link below via email or IM

Copy

Present to your audience

Start remote presentation

  • Invited audience members will follow you as you navigate and present
  • People invited to a presentation do not need a Prezi account
  • This link expires 10 minutes after you close the presentation
  • A maximum of 30 users can follow your presentation
  • Learn more about this feature in our knowledge base article

Do you really want to delete this prezi?

Neither you, nor the coeditors you shared it with will be able to recover it again.

DeleteCancel

Make your likes visible on Facebook?

Connect your Facebook account to Prezi and let your likes appear on your timeline.
You can change this under Settings & Account at any time.

No, thanks

Illumina genome sequencing

28th June 2012 - Bioinformatics course @ BaseClear
by

Adalberto Costessi

on 3 April 2014

Comments (0)

Please log in to add your comment.

Report abuse

Transcript of Illumina genome sequencing

Amount & concentration
Broad-Range (Qubit)
Picogreen
don't use Nanodrop!
Integrity

Purity
HiSeq 2000 (v3):
2 independent flowcells
up to 150 mln reads per lane
up to ␣100␣ bases readlength
single read and paired-end read sequencing
Product specialist
Next Generation Sequencing
ADALBERTO
COSTESSI
Illumina Sequencing
1953
Discovery of dsDNA
1965
First nucleic acid
sequenced:
E. coli tRNA
1977
Sanger sequencing
&
First sequenced genome:
PhiX 174 (5,4 kb)
Start of Human
Genome Project
H. influenzae
first sequenced
free-living organism
History of DNA sequencing
Illumina sequencing
Advantages & disadvantages:
Highest throughput (max 600 GB/run)
High-level sample multiplexing
Max read length PE 100 cycles
"Long" sequencing times (dependent on read length)
Lowest cost per base

On the market in 2006/7
Sequencing␣-by-␣synthesis approach␣–
1 Flowcell with 8 independent lanes
GAIIx: up to 30-40 mln reads per lane
Genome sequencing
GA (Solexa)
HiSeq 2000
Next-next generation (?)
Data output:
Sequence data in FASTQ
Error rate ~1%: only substitutions, no indels
Homopolymeric stretches are handled well
Problems with high AT or GC rich regions
http://www.qbi.uq.edu.au/images/genomics/genomics1.jpg
http://seq.molbiol.ru
http://microarrays.nki.nl/uploads/images/flowcells.jpg
Applications:

Genome sequencing
de novo
resequencing

Targeted resequencing
Exomes
Target genes/loci

Metagenomics
16S
shotgun

Transcriptome
mRNA-seq
small RNA

Epigenetic
ChIP-seq
5-mC and 5-hmC (EpiQuest)
Paired-end
Mate pair
100-500 bp
2-5 kb
2 ug
10 ug
Insert size
Input DNA
gDNA sample requirements
Samples
are delivered at BaseClear
Project number assigned
E-mail confirmation
Registration on logbook @ reception
Project
setup
Registration in LIMS system
Samples
are brought to the lab
Project
control and planning
Library preparation
Sequencing
Data
QC
Assembly,
scaffolding &
gap closure
Genome
released
Sample
QC
Library
QC
Update
to customer
Update
to customer
contig 1
contig 2
scaffold
PE
MP
2-5 kb
300 bp
PacBio
Nanopores
True single molecule w/o PCR amplification
Optical detection (4 fluorochromes)
Very long reads:
median of 2-2,5 kb
up to 15 kb
High error rate ~13-15%
Very fast (2h per run)
8 SMRT cells per run & ~100 MB/SMRT cell
Input DNA: 5-10 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.
Oxford Nanopore
Genia technologies
Nabsys inc
Base 4 innovations Ltd
Quantapore Inc
...
Fast & looooong reads
but
higher error rates
Biological pore
Electrical detection
Announced:
up to 100kb reads
4% error rates
1
2
4
3
http://www.clker.com/cliparts/1/a/2/e/1197085885105139227CrazyTerabyte_Book.svg.med.png
http://www.neb.com/nebecomm/productfiles/3313/images/E7335c_v1_000005.jpg
Adalberto Costessi
Product Specialist NGS
BaseClear

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

1998-2003: MSc Medical Biotechnology, University Trieste (I)

2004-2005: European Space Agency (NL)
Trainee and P.I. of "Bone Proteomics Experiment"

2005-2010: PhD Molecular Biology, Henk Stunnenberg lab, Radboud University Nijmegen (NL)

2011-present: Product Specialist NGS, BaseClear BV, Leiden (NL)
http://www.nature.com/nature/journal/v458/n7239/full/nature07943.html
http://www.genome.gov/sequencingcosts/
http://www.ncbi.nlm.nih.gov/genbank/genbankstats-2008/
http://www.linkedin.com/in/costessi
Drew Sheneman, New Jersey -- The Newark Star Ledger
http://www.youtube.com/watch?v=l99aKKHcxC4
Yes
Barcoding
Yes, BaseClear protocol
http://www.pacificbiosciences.com
28th June 2012
Full transcript