amplicon sequencing by ngs methods · amplicon sequencing by ngs methods jean-françois martin...
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Amplicon sequencing by
NGS methods
Jean-François Martin
Centre international d’études supérieures en sciences agronomiques
www.supagro.fr
Centre de Biologie pour la Gestion des Populations
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By Jean-François Martin
Goals and expectations
The aim of this discussion today : review the different options to
massively sequence amplicons through NGS technology and show
some current work on adapting protocols.
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Why sequence amplicons ?
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Amplicon sequencing
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Applications overview
Applications of next generation sequencing in molecular ecology of non-model organisms, Heredity, 2011
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Most commonly used applications:
• Barcoding
• Metagenomics (including metabarcoding)
• Phylogeny
• Microsatellites genotyping (seriously?!)
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Amplicon sequencing
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Most of those applications could also be addressedby capture methods
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Amplicon sequencing
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The applications will require different analysismethods. This is important in designing the project.
Think about the output information and format youwill use for further analysis
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Amplicon sequencing
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Example among others : environmental barcoding
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Amplicon sequencing
Sampling
DNA
extraction
ResultsBioinformatics pipeline
High throuput sequencingPCR
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Amplicon sequencing
Goals and applications
Characterization of the environmental species diversity
Characterization of parasites
Characterization of diet
Trophic network
Microbiome analysis
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It is required to setup a simple and efficient methodology to acquire data so it makesamplicon sequencing accessible to anyone
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Amplicon sequencing
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What technology should we choose for sequencingamplicons ?
It depends !
but in any case error rate should be as low as possible
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Amplicon sequencing
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Preliminary testing of pacbio RS on long amplicons
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Amplicon sequencing
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Amplicon sequencing
• 100k sequences, including 19-
21k ccs
• Up to 17k bases / sequence at
the time (march 2014)
• Highly variable quality from one
run to the next
• 15% eror rate on controls
Preliminary testing of pacbio RS on long amplicons
Today on a Pacbio RS II, 15kb median, 40kb max
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Preliminary testing of pacbio RS on long amplicons
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Amplicon sequencing
98% of the css fragments are of the correct size (658pb)
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Preliminary testing of pacbio RS on long amplicons
• 42% of quantitative variation between technical replicates
• Majoritary sequence (17% in average) always correspond to the expected
• Random error based correction always recovers the correct sequence
• The number of errors/sequence never exceeds 2 (out of 658 bases)
• When mixed samples occur, the reproducibility of ratios is very low
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Amplicon sequencing
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Preliminary testing of pacbio RS on long amplicons
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Amplicon sequencing
Conclusion : SMRT is very useful for barcoding
long amplicons (658pb) but not usable in
environmental applications.
Although compared to sanger it is still expensive
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Designing amplicon sequencing strategy withMiseq sequencing
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Amplicon sequencing
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Designing amplicon sequencing strategy withMiseq sequencing
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Amplicon sequencing
Goal : simultaneously sequence up to thousands of
amplicons
• How many sequences for each amplicon ?
• How do I multiplex the amplicons ?
• What are the limits of the technology ?
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Designing amplicon sequencing strategy withMiseq sequencing
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Amplicon sequencing
How many sequences for each amplicon ?
• What is the expected variation for an amplicon ?
• How many sequences to validate a variant ?
• Also means that all amplicons are equitably represented
in the sequencing run and all sequences are usable
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Designing amplicon sequencing strategy withMiseq sequencing
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Amplicon sequencing
How do I multiplex the amplicons?
What multiplexing level do I need ?
What are the built-in multiplex limits for Illumina ?
How do I combine amplification and sequencing ?
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Designing amplicon sequencing strategy withMiseq sequencing
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Amplicon sequencing
What are the limits of the technology?
Are there artefacts that matter for my application ?
How many amplicons can I sequence in a given period of
time ?
What are the costs (direct and indirect) for the different
strategies ?
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The 1-step tagged PCR + library strategy
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Amplicon sequencing
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The 1-step tagged PCR + library strategy
• Amplify the target with tagged primers
• Pool the amplicons by marker / size …
• Prepare a library for each pool
• Sequence (Miseq v3)
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Amplicon sequencing
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Step 1 : amplification process
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Amplicon sequencing
Primer
Percentage for each base
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The 1-step tagged PCR + library strategy
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Amplicon sequencing
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The 1-step tagged PCR + library strategy
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Amplicon sequencing
empty
negative
Positive but
different
Plate design
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The 1-step tagged PCR + library strategy
How many tag combinations and how many indexes ?
In other words should I maximise the number of PCR ?
For reference, 1 index / library -> 90€ + handling effort
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Amplicon sequencing
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Step 2 : combining amplicons as pools
How do I normalize the amplicons before pooling ?
old-school method : they all look the same on my gel, I pool them as is.
refined old-school : I create a couple of categoriesaccording to intensities on gel and pool differentvolumes accordingly
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Amplicon sequencing
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Step 2 : combining amplicons as pools
How do I normalize the amplicons before pooling ?
the « what I can I do best for my precious ?» strategy, uses a sequalprep kit to retain known quantity of amplicon (25ng) then pool them
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Amplicon sequencing
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Step 2 : combining amplicons as pools
It is advised not to mix different size amplicons as it wouldfavor the shortest ones in the process (PCR if anyduring the library and sequencing).
1 pool = 1 library
Should I do replicates ?
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Amplicon sequencing
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Step 2 : combining amplicons as pools
Common problems are linked to successfully amplify a widerange of species with an equal efficiency
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Amplicon sequencing
Improve
efficiency
Universal primers Primers cocktail
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Step 2 : combining amplicons as pools
Gel purification of the pools
Qualifying the pools on bioanalyzer
Quantifiying the pools with what is convenient (nanodrop)
Normalize pools to 0.4ng/µL (20 ng in 50µL)
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Amplicon sequencing
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Step 3 : building libraries for each pool
This protocol uses part of the Trueseq nano LT kit (4h)
1. End-repair to make the amplicons blunt-ended.
2. A-tailing to allow further ligation
3. Ligate the illumina adapters (holding indexes)
4. Enrich in P5-P7 libraries through amplification
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Amplicon sequencing
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Step 3 : building libraries for each pool
When the libraries are built, I need to qualify them usingthe bioanalyzer
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Amplicon sequencing
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Step 3 : building libraries for each pool
The last step is to quantify the libraries with qPCR usingadapter sequences as primers.
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Amplicon sequencing
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Step 4 : Miseq sequencing
Preparing the sample (pooled libraries) involves dilution and denaturation in NaOH.
Setup of the sequencer takes around an hour (warming)
The run is 46 hours long
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Amplicon sequencing
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Step 4 : Miseq sequencing
What can go wrong at the sequencing step?
• Overclusterization
• Bad quality
• Door opened..
• Road construction…..
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Amplicon sequencing
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Two other wet lab strategies 2-steps PCR
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Amplicon sequencing
Advantages : multiple markers, universal and easy
Drawbacks : 2 steps PCR costs, biases and contamination issue
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Two other wet lab strategies : 1-step PCR
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Amplicon sequencing
Advantages : 1 step PCR, no primer sequencing
Drawbacks : add custom primers into the sequencer cartridge, only 16S for now
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comparing wet lab strategies
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Amplicon sequencing
Few markers and
< 1728 samples
Metabarcoding
16S (bacteria)
Multiple projects,
> 1728 samples
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Amplicon sequencing
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Amplicon sequencing
Data analysis, from raw data to usable data
General steps:
1. quality assessment
2. filtering using quality
3. contig of paired reads
4. demultiplexing the markers if relevant
5. demultiplexing the amplicons
6. characterizing variants
7. Aligning variants if necessary
8. assigning to a reference library
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Amplicon sequencing
Data analysis, from raw data to usable data
1. quality assessment (Galaxy)
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Amplicon sequencing
Data analysis, from raw data to usable data
2. Filtering the reads (Galaxy)
3. Contig the paired reads (Mothur) and prepare a
fasta file
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Amplicon sequencing
From now, everything is done with
Could also be done with
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Amplicon sequencing
The pipeline
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Amplicon sequencing
The sequences alignment interface (if needed)
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Amplicon sequencing
Preparing reference libray
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Part 1
Part 2
Part 3
Part 4
Part 5
By Jean-François Martin
Amplicon sequencing
Assignment to the library
Assign to the first
common ancestor if
similar at 95% threshold
Assign to the
closest species,
even if far away
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Part 1
Part 2
Part 3
Part 4
Part 5
By Jean-François Martin
Amplicon sequencing
Exploring results
Detected taxa, samples
and sequences
List of samples
(multiple sel.)
Graphical results
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Part 1
Part 2
Part 3
Part 4
Part 5
By Jean-François Martin
Amplicon sequencing
Concluding remark about future directions
Improving PCR of
alternative approaches
Work on the scalability of
bioinformatics solutions
Be aware of the
continuous evolution
of thechnologies
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By Jean-François Martin
Acknowledgements
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Centre international d’études supérieures en sciences agronomiques
www.supagro.fr
Centre de Biologie pour la Gestion des Populations