This document summarizes the work of Hans Jansen and Christiaan Henkel with long read nanopore sequencing. They have sequenced several genomes including carp, eel, king cobra, and Agrobacterium using MinION. Their longest reads were 120 kbp and 93.5 kbp. They also established the MinION Access Program to improve genomes by resolving repeats. As part of this, they formed the MinION Analysis and Reference Consortium to standardize protocols and understand variability between labs. Their work with the E. coli genome demonstrated sources of variation in read counts, lengths, and alignments between labs.
1. Scaffolding using long nanopore reads
and more
Hans Jansen
Christiaan Henkel
senior scientist
2. Dutch SME at Bioscience Park in Leiden, the Netherlands
• High throughput drug screens, and toxicity assays in zebrafish larvae
• Fish fertility (eel, pike perch, sole) to aid sustainable aquaculture
• Sequencing (genomes, transcriptomes)
• Bioinformatics
ZF-screens B.V.
3. Genome projects
Common carp (Cyprinus carpio)
High troughput screening model
Genome and transcriptomes
European and Japanese eel (Anguilla anguilla and Anguilla japonica)
Completing the life cycle in aquaculture
Genome and transcriptomes
King cobra (Ophiophagus hannah)
Evolution and toxins
Genome and transcriptomes
But the quality of these genomes can be improved
4. But MAP is much more. It is about being a community and a playground to test new
applications. As Gordon Sanghera (CEO of ONT) said "MAP will never end. There will
always be a MAP“.
So if you think you're application can benefit from nanopore sensing then come join
MAP and play with us.
Visible as a web portal with information from ONT and social media like system with
blog possibilities, comment, likes, and a forum to ask advice.
MinION Access Program
5. We entered when MAP started.
Our first MinION arrived in April 2014 and the first kits in June.
Since then run 30 Flow Cells.
MAPpers competition
Topped the leaderboard on read length and yield so we now have three MinION's.
MinION Access Program and ZF-genomics
8. Cheap short read sequencing technology has been used to generate many draft genomes
repeatunique sequence in unique sequence out
Draft genomes made with short read data suffer from a fundamental problem.
Reads that are shorter than the length of a repeat can’t connect the unique sequence in with
the unique sequence out
Genomic sequences
Short reads
9. repeatunique sequence in unique sequence out
Long reads can help to resolve repeat area’s in the assembly graph
And the resulting contigs will now look like this:
Untangle
10. 1. Short read correction Quake (not for small genomes)
2. Short read assembly Velvet
3. MinION read alignment to Velvet contigs LAST
4. Link filtering and contig tiling Untangle script
5. Path detachment around repeats Untangle script
6. Bubble popping Untangle script
7. Delete unconfirmed connections Untangle script
8. Contig extraction Untangle script
Assembly and scaffolding strategy
Task Software
11. Agrobacterium strain NCPPB 1771
Agrobacteria are the cause of crown gall
disease, a tumorous growth of plant tissue.
Agrobacteria transfer part of their (plasmid)
DNA to their host and this feature is used
widely in plant research to genetically
modify plants.
Agrobacteria have two chromosomes, and
carry several plasmids. This strain also
carries active transposons.
13. • Alignment: LAST with optimized settings
• Links: alignment filtering and contig tiling
• 7328 reads aligned to contigs
• 438 reads aligned to multiple contigs
• 585 links between contigs
• 13158 reads on R6 and R7 chemistry
• 73.8 Mb total yield (template and 2D)
• 5–85970 nt length, typical ~12 Kb
MinION sequencing and scaffolding
16. MinION Analysis and Reference Consortium
MARC is a consortium within MAP that seeks to establish sources of variation,
optimize protocols and analysis.
It is open science. Data is shared in the consortium and will be made available
through ENA.
~100 people have signed up. ~7 experimental groups and ~4 analysis groups
are actively working.
Managed by weekly TC.
17. Different phases in MARC
Phase 1 is about being as standard as possible and establish variation in the
system and between sites.
This is done by 5 labs in the Netherlands, UK (2), USA ( east and west coast).
Phase 2 is all about tweaking the protocol. Things like DNA isolation, shearing
(or not), running scripts, DNA modifications will be addressed in this phase.
Phase 3 is about examples of applications.
MinION Analysis and Reference Consortium
18. MinION Analysis and Reference Consortium
In phase 1 the 5 participating labs received Escherichia coli str. K-12 substr. MG1655.
Performed DNA isolation, library prep, and sequencing according to a detailed protocol.
Per lab a total of 4 libraries with 2 different kits were prepared and run.
This provides a excellent data set to understand sources of variance in ONT data.
22. With the data of the first 10 runs analyzed we can already see that read length has a
stronger lab effect than base pair identity to the reference.
Another set of 10 phase 1 runs is currently being analyzed and will give a clearer
picture on variability.
Experiments for phase 2 will start shortly, while in parallel phase 3 experiments and
analysis are being done.
Conclusions and perspectives
23. The king cobra genome
Rapid expansion of the 3 FTx gen family in
the king cobra
24. London Calling 2015
Highlights from Clive Brown’s talk
• Improvements to the basecaller . There’s still room for improvement.
• Read until (and barcoding).
• Fast mode on the MinION MkI (500 bp/sec instead of 30)
• New 3000 channel ASIC with crumpet chip design to separate ASIC and fluidics part.
• MinION MkII and PromethION will have this new ASIC.
• Library prep on beads to reduce amounts of DNA needed (lower ng to pg).
• Direct RNA sequencing.
• Simplified sample preparation and VolTRAX.
• Pricing will be “pay as you go”. Initial payment for hardware include some hrs sequencing.
• MkI $270 and 3 hrs sequencing (~3 Gbp in fast mode).
25. Acknowledgements
Prof. Dr. Paul Hooykaas, Leiden University
Christiaan Henkel
senior scientist
Leiden University
Ron Dirks (CEO of ZF-screens B.V.)
All members of the MARC consortium
Ewan Birney, EMBL-EBI
Justin O’Grady, UEA
Sara Goodwin, CSHL
David Buck, WTCHG Oxford
Vadim Zalunin, EMBL-EBI
Miten Jain, UCSC
Matt Loose, Nottingham
Jared Simpson, OICR, Toronto
Notas del editor
Excuse me if I may sound like a ONT salesperson, but the truth is nanopore sensing is a very powerful method to measure many different things and it will show up on many different places in your life over the next decade or two.
