Periodic Reporting for period 1 - FuseSeq (Single molecule reconstruction for high-throughput, short-read sequencing technologies)
Reporting period: 2022-06-01 to 2023-11-30
The fundamental role of large-scale alterations in nucleic acids, such as structural variants and splice variants, in cancer and neurodegenerative diseases is getting clearer. However, due to technological limitations of current advanced short-read sequencing technologies and third generation long-read sequencing technologies full molecule reconstruction in a high throughput manner is still not possible. To fully understand the contribution of these changes in nucleic acid sequence a technology is needed that would be able to determine the sequences of large numbers of individual molecules of an unprecedented length independent of their sequence complexity and the presence of repetitive elements in them.
To address this problem this project aimed to test the feasibility of a method that would bridge the gap between long reads and high throughput sequencing by developing a fragment labelling system compatible with high-throughput short-read sequencing technology. The labelling system consist of tags that can be incorporated into target nucleic acid sequences using a hyperactive transposase mutant and can be subsequently used to create fragments from the target molecule while retaining information about the fragments' original position in the target molecules, thus allowing the reconstruction of the sequence of the original, individual target molecules from them.
To address this problem this project aimed to test the feasibility of a method that would bridge the gap between long reads and high throughput sequencing by developing a fragment labelling system compatible with high-throughput short-read sequencing technology. The labelling system consist of tags that can be incorporated into target nucleic acid sequences using a hyperactive transposase mutant and can be subsequently used to create fragments from the target molecule while retaining information about the fragments' original position in the target molecules, thus allowing the reconstruction of the sequence of the original, individual target molecules from them.
As part of project a protocol has been finalized to produce probes with sufficient yield for downstream applications that contain all segments required for the intended function of the probes.
Several chemical strategies have been tested suitable for fragment generation and number of suitable strategies have been identified that successfully mediated chemical cleavage of the probes. As these approaches would require extra steps and introduction of new substances into existing sequencing library protocols, they might interfere with them. To avoid this, an alternative strategy has also been tested that utilizes chemical modification that results in the generation of fragments in parallel to the routine sample amplification steps found in sequencing library preparation protocols. This fragmentation strategy allows the usage of our probes in existing sample preparation protocols for sequencing library generation with minute alterations and thus will make the final method be easily includable as part of other commercially available sample preparation approaches for next generation sequencing increasing the future accessibility of the method for end-users.
Finally, we tested the insertion of probes into nucleic acid target molecules via transposase. We have confirmed the insertion of them using commercially available formulation of the transposase enzyme, however the achievable insertion frequency was lower than required for our final application. Results with an in house produced transposase mutant indicated insertion of the probe allowing the possibility to finetune the insertion frequency to reach the required levels for the final application.
The project has thus successfully demonstrated the feasibility of all the crucial steps required for method to be applied to complex samples.
Several chemical strategies have been tested suitable for fragment generation and number of suitable strategies have been identified that successfully mediated chemical cleavage of the probes. As these approaches would require extra steps and introduction of new substances into existing sequencing library protocols, they might interfere with them. To avoid this, an alternative strategy has also been tested that utilizes chemical modification that results in the generation of fragments in parallel to the routine sample amplification steps found in sequencing library preparation protocols. This fragmentation strategy allows the usage of our probes in existing sample preparation protocols for sequencing library generation with minute alterations and thus will make the final method be easily includable as part of other commercially available sample preparation approaches for next generation sequencing increasing the future accessibility of the method for end-users.
Finally, we tested the insertion of probes into nucleic acid target molecules via transposase. We have confirmed the insertion of them using commercially available formulation of the transposase enzyme, however the achievable insertion frequency was lower than required for our final application. Results with an in house produced transposase mutant indicated insertion of the probe allowing the possibility to finetune the insertion frequency to reach the required levels for the final application.
The project has thus successfully demonstrated the feasibility of all the crucial steps required for method to be applied to complex samples.
The project has demonstrated the feasibility of the fundamental steps required for the proposed method to be applied to sequence reconstruction: production of probes carrying all required functional elements with high enough yield for downstream application, fragmentation strategy using easily incorporable chemical modification that would allow easy integration of the method into existing sample processing protocols and finally the insertion of the probes into nucleic acid target molecules using transposase.
For the method to be applicable for complex samples and permit single molecule reconstruction further research is needed to achieve high enough incorporation rates required for generation of fragment sizes compatible with existing short-read sequencing methods. The results obtained using an in-house produced transposase mutant shows promise to address this issue and thus further future work will focus on pursuing this strategy and the adaptation of the method to different short-read sequencing methods.
For the method to be applicable for complex samples and permit single molecule reconstruction further research is needed to achieve high enough incorporation rates required for generation of fragment sizes compatible with existing short-read sequencing methods. The results obtained using an in-house produced transposase mutant shows promise to address this issue and thus further future work will focus on pursuing this strategy and the adaptation of the method to different short-read sequencing methods.