How to produce a perfect sequence

Hana’s latest enthusiasm is the utilization of optical maps done by BioNano Genomics technology (IRYS platform) to increase the quality of chromosome-based reference sequences.

“With optical map in hand, we can solve many problems. We can verify if BAC clones were correctly assigned to the physical map, identify misassemblies, and see gaps. A large number of markers are needed to anchor contigs of physical maps to chromosomes. Despite a big effort, many contigs, especially the short ones, usually remain non-anchored. With this tool we are even able to position contigs consisting of only three BAC clones. Now we know precisely where on the chromosome they are located, how they are oriented, or if we have any gaps in the physical map. For me, this is the way to produce a nearly perfect sequence.”

The tool provides a picture of the reality “because we can see the distribution of a short sequence motif along DNA stretches which are hundreds kb to megabases long. This is very useful to validate a sequence assembly.” Optical maps also have potential to reveal the presence and organization of large regions of tandem repeats, which are particularly difficult to resolve during the assembly of sequence data.

Another application of the technology, currently used in human genomics, is the study of structural and copy number variation between various genotypes by identifying deletions, insertions, inversions, and duplications. “That is where we would like to go in the future. In our pilot study, which was a part of a resistance gene cloning project, we compared our region of interest on the 7DS chromosome arm in two varieties of wheat with the aim of learning if the Chinese Spring sequence, which we have in hands, covers completely the region in our resistant line.”

Hana’s team is currently using the optical map technology to validate the sequence assembly of chromosome arm 7DS. Also, the team of Rudi Appels in Australia is using the 7A optical map generated at the IEB for their sequence assemblies and optical maps for the 7B chromosome (coordinated by Odd-Arne Olsen in Norway) will be completed soon.

“If there is an interest, we can produce optical maps for all wheat chromosomes. It’s feasible.

12 years in the making

Hana, as part of Jaroslav Doležel’s team, has been involved with the IWGSC from the start “thanks to Catherine Feuillet who realized that the chromosome strategy was the best one to tackle the wheat genome.”

Even before the official birth of the IWGSC in 2005, she had optimized the procedure of preparation of high molecular weight DNA from flow sorted chromosomes and produced the material that was used by Jan Šafář, in collaboration with Boulos Chalhoub, to construct the first BAC library of a wheat chromosome – 3B – in 2002. With Jan, she then optimized the procedure and started working on the production of BAC libraries for the 21 wheat chromosomes.

The whole procedure, which comprised construction of 36 chromosome – or chromosome-arm – specific BAC libraries, took 12 years. The construction of one library typically involved 10 highly specialized and experienced persons. “Once you have robust protocols, a pipeline and experienced people, it’s easy. We have a great experienced team and this is our key to success.”

Sequencing project

Concurrently, Hana worked on other projects including the sequencing of chromosome arm 7DS.

“Having a sequencing project was a big challenge for me because I am a wet lab person, not a bioinformatician. The physical map was manageable, but sequencing and sequence assembly was too big of a challenge. In the beginning, we did not have a sequencing facility or experience. So, we were pleased that the group of Dave Edwards at the University of Western Australia in Perth, with whom we previously collaborated on survey sequencing wheat chromosomes and who had experience with genome sequencing projects, agreed to become involved. They sequenced BAC clones from physical map of 7DS and assembled the sequences for us.”

Hana and her team now have a draft assembly of the 7DS chromosome arm ready and they are approaching the final stage of constructing the pseudomolecule. She is looking forward to the upcoming IWGSC sequencing calls and PAG workshops to discuss this new challenge with other teams.

Collaboration is expected

“It is great that we can interact with other people in the Consortium during conference calls where people who are advanced in their sequencing projects or test new approaches share their experience or present their newly developed resources. I was also very glad to know that the Standards & Protocol workshop at PAG 2016 will focus on pseudomolecule assembly. This is something that became a very hot topic for us and other labs that have reached this stage. It will be good to coordinate these activities so that all chromosomal sequences reach the same standard.”

“This is for me the biggest merit of the Consortium. I consider producing all the BAC resources and, eventually, the optical maps our contribution to the community. On the other hand, I am learning from others how to make things better. The Standards & Protocols workshops are usually the highlights of the PAG meetings for me because in contrast with other workshops where people present their best results, here you hear about the problems. This is more inspiring."

Publication date: 10/12/2015