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Bacterial genome analysis using ...

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How to close
your bacterial
genome using
Oxford
Nanopore
sequencing
technology?

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Bacterial genome analysis using ...

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Next generation sequencing (NGS)


has given many laboratories
access to high-throughput
bacterial genomic research. The
number of bacterial genomes
sequenced is vertiginously
increasing, yet the fraction of
completely closed (single contig)
assemblies is extremely small
compared to the number of
generated (incomplete) draft
genomes. The explanation is
rather easy: the costs to generate a
draft genome using short read
technologies is manifold less than
the costs associated with long read
third generation sequencing

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Bacterial genome analysis using ...

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technologies such as offered by


Pacific Biosciences. And costs
should be seen in a broad sense:
from the costs of chemicals and
laboratory personnel, to the
expenses of the sequencing
platform (around $700.000 for the
Pacific Biosciences PacBio RS II
platform) and last but not least
the data analysis!
The PacBio RS II platform is available
for a few years now and uses SMRT
technology (Single Molecule Real
Time sequencing) to generate long
sequence reads of up to 30 kbp. It
offers an advantage over other

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technologies in that it can sequence


individual DNA molecules by
recording the incorporation of
fluorescent labelled nucleotides in
real-time. The sequence bias of the
PacBio platform is relatively high
(approximately 10%), but in order to
improve sequence quality the PacBio
system can ligate adapters at both
ends of each DNA molecule to form a
continuous DNA circle. This allows
each DNA molecule to be sequenced
through multiple passes to build a
more accurate consensus read. The
technology has not yet matched
second generation platforms in
terms of sequence quality but has

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Bacterial genome analysis using ...

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proven useful in various applications


including contig scaffolding.
In the past years we have put much
effort in creating workflows which
involves a hybrid analysis approach
where both Illumina short reads and
PacBio long reads are combined to
create closed genomes of extremely
high quality. Not only lab protocols
are optimized, also a full data
analysis pipeline, called SSPACELongRead (Boetzer and Pirovano,
2014) has been developed: whereas
the Illumina technology typically
generates genome assemblies
comprising 20500 contigs (mainly

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due to the presence of repetitive


elements which are larger than the
short read length), the addition of
PacBio long reads generally leads to
a fully closed bacterial genome (one
contig per chromosome and possible
plasmids).

Will we be
able to easily
close
bacterial
genomes in

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Bacterial genome analysis using ...

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a
high-throughput
manner
when read
lengths go
beyond
100Kbp
though with
an error rate
of
approximately
10%?

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Bacterial genome analysis using ...

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But change is coming! The latest


long read sequencing technologies,
in particular that of Oxford Nanopore
Technologies, have the potential to
further ease the process and
importantly breakdown the
analysis costs. So-called USB-stick
DNA sequencing is promised to have
an enormous strong impact on
genomic research as the library
preparation and sequencing steps
will gradually become only a fraction
of the costs of current long read
sequencing technologies. As a
matter of fact, Oxford Nanopore
Technologies started in 2014 with an
early access program of their first

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Bacterial genome analysis using ...

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robust sequencer (The MinION


Access Program, MAP). Likewise the
PacBio system, it delivers long read
real-time sequencing of individual
molecules. It is the first commercial
nanopore-based (rather than
synthesis-based) sequencer. The
MinION is quite small and
portable, having the size of a USB
stick. The current throughput is still
limited but quickly increasing to
several Giga-bases per run! And this
isnt yet all: in 2015 the
PromethION will be offered in an
early access program, which is a
small benchtop system for high
throughput real time biological

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Bacterial genome analysis using ...

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analyses and allowing larger sample


numbers. This will give a further
boost to the technology and make it
suited for sequencing 96-wells plate
in no-time!
Meanwhile different solutions are
being developed for easy and on-site
library preparations, can it be a
matter of time before this becomes
routine work? And when will these
solutions become available for a
large audience (and not only through
an advance access program)? And
will we be able to easily close
bacterial genomes in a
high-throughput manner when read

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Bacterial genome analysis using ...

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lengths go beyond 100Kbp though


with an error rate of approximately
10%? It seems that the revolution
has started and no wonder the
participants of the recently held
London Calling meeting, where
different partners of the MinION
advanced access program presented
their findings, returned home with a
huge smile on their face (and a
personalized MinION). But there
are still some hurdles to take, also at
the bioinformatics side
So what about the development of
appropriate analysis tools for long
read sequencing? BaseClear

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Bacterial genome analysis using ...

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bioinformaticians are at the


forefront of data analysis and in
particular the published genome
finishing tools SSPACE Standard and
GapFiller are largely recognized by
the field which is underscored by a
huge number of citations! The latest
assembly plugin developed
internally by the BaseClear
Bioinformatics team, SSPACELongRead (Boetzer and Pirovano,
2014,http://www.biomedcentral.com
/1471-2105/15/211), is well capable
of merging the short-read Illumina
data together with long-read PacBio
or Nanopore data into one hybrid
assembly to completely close

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Bacterial genome analysis using ...

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http://www.baseclear.com/blog/ba...

genomes. Currently also this latter


tool is becoming more and more
recognized by the community. As
such recently SSPACE-LongRead is
used by a group of researchers who
evaluated the utility of the MinION
sequencing system of Oxford
Nanopore for scaffolding bacterial
genomes by comparing MinION
sequencing data for two Francisella
genomes: the findings of Karlsson
and co-workers were published in
Nature Scientific Reports
(http://www.nature.com/srep/2015
/150707/srep11996
/full/srep11996.html). SSPACELongRead was used together with

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Bacterial genome analysis using ...

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BLASR (Chaisson and Tesler, 2012) to


scaffold the contigs using the
MinION reads. The MinION reads
could correctly join all of the contigs
to a complete chromosome.
Although first successful results
using Oxford Nanopore technology
are now published, making reliable
assemblies of bacterial (or other)
genomes remains challenging. The
right preparation, coverage,
software, parameters, etc. need to
selected in order to obtain best
quality de novo assembly results. For
this the right bioinformatics
expertise and experience is needed

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Bacterial genome analysis using ...

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as there are many pitfalls. Oxford


Nanopore technology will be a good
alternative for PacBio sequencing
and in time might omit the use of
Illumina sequencing for genome de
novo sequencing projects. But it will
take some more time before
bacterial genome de novo
assemblies will be a real piece of
cake and can be applied by any
researcher in any laboratory.
Meanwhile you can be sure that
BaseClear bioinformatics are taking
the challenge to create analysis
pipelines and assembly tools that
are at the forefront as new
sequencing technologies enter the

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Bacterial genome analysis using ...

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scene!

July 2015 by Walter Pirovano Director bioinformatics BaseClear


B.V.

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