Circular sequencing by Geno1® technologies

The circular ssDNA molecules containing the Nicking Loops are either achieved by targeting in the Bridge Capture™ (see How does GapFill work in the Bridge Capture™?) or with non-targeted Nicking Loop™ conversion & indexing (seequestion How is the Nicking Loop™ conversion & indexing done?). These serve as a template for the linear rolling circle amplification (RCA) reaction (Figure 1 step 1). RCA produces a long concatemeric molecule containing multiple adjacent linear copies of the original circular template (see question How does the rolling circle amplification (RCA) work in Bridge Capture™ and Nicking Loop™?). A long linear molecule generated by RCA contains sequence structures that facilitate the formation of local hairpin loops at fixed positions (Figure 1 step 2). Each segment, spanning from the complete loop structure to the start of the next adjacent loop, represents one full cycle around the circular template. The double-stranded stem of each loop includes a recognition site for a specific nicking endonuclease enzyme, able to cleave only one strand of the stem sequence. This targeted digestion acombanied with mild heating cuts the long concatemeric molecule into multiple pre-defined DNA fragments, each representing a linear copy of the original circular template (Figure 1 step 3). The ends of these fragments contain complementary sequence motifs, enabling them to self-anneal. With the addition of a ligase, the fragments can be re-circularized into complementary copies of the original template (Figure 1 step 4). These circular molecules can serve either as templates for further amplification cycles or as ready-to-use sequencing libraries with circular sequencing platforms (see What are the outputs of Bridge Capture™ and Nicking Loop™ processes?).

The Nicking Loop™-converted circular DNA molecule is constructed from a DNA template, a bridge oligomer, a Nicking Loop, and Y-adapters. The process involves the following steps: 1. Template Preparation: The template, which can be any unknown genomic DNA or linear library molecule, is ligated with Y-adapters at its ends to create binding sites for a universal bridge oligomer complex. 2. Circularization: The bridge oligomer complex binds to both Y-adapter ends of the template, forming a circular DNA structure. A Nicking Loop is also introduced here to the construct, enabling an early stage sample indexing. 3. Ligation: Nicks in the circular structure are sealed, completing the formation of a single-stranded DNA (ssDNA) circle. Excess non-circularized molecules are then removed using exonucleases. 4. Amplification: The intact circular molecules are amplified using rolling circle amplification (RCA). For further details, refer to the question: How the Nicking Loop™ copying is done?

Yes, optionally. Bridge Capture™ as well Nicking Loop are designed to be compatible with UMIs (see question What is a UMI and how are they implemented in the data processing?). In Bridge Capture™ UMIs are located in the probe arms (see question What is a probe?), whereas in Nicking Loop™ UMIs are incoporated to the loop structure (see question Can a sample be indexed with Nicking Loop™?). UMIs are useful for ultrasensitive quantitative applications, such as detecting low allele fractions from cfDNA. However, UMIs may not be necessary for the applications where simple presence or absence quantification is required.

Yes. The loop structure of the Nicking Loop molecule is designed to accomodate pre-determined index and/or Unique Molecular Identifier (UMI) sequences. During the Bridge Capture™ target capture (see question How does the targeting work in the Bridge Capture™?) and gap fill reactions (see question How does GapFill work in the Bridge Capture™?) Nicking Loops are attached to the probe complexes. In the Nicking Loop™ conversion & indexing the Nicking Loop is introduced in the circularization step (see question How is the Nicking Loop™ conversion & indexing done?). Both workflows generate indexed circular ssDNA library molecules. The incorporation of these index sequences and UMIs ensures accurate sample identification and sensitive molecular tracking during downstream analysis.

Yes. The Nicking Loop™ conversion & indexing (see question How is the Nicking Loop™ conversion & indexing done?) enables the creation of circular single-stranded DNA (ssDNA) whole-genome libraries, facilitating comprehensive whole-genome sequencing.

Bridge Capture™ and Nicking Loop™ enable the production of two distinct types of DNA libraries: linear and circular libraries. Circular libraries are generated either from the primary gap-fill reaction of the Bridge Capture™ workflow (see question How does GapFill work in the Bridge Capture™?), converting any DNA into circular libraries (see question How is the Nicking Loop™ conversion & indexing done?) or through the Nicking Loop™ copying process (see question How the Nicking Loop™ copying is done?), which directly amplifies circular single-stranded DNA (ssDNA) molecules. These circular ssDNA molecules can function either as templates for additional amplification generations or as ready-to-use sequencing libraries. The size of the circular libraries typically ranges from 250 to 400 bases. Linear libraries are produced by PCR amplification. Template for amplification is either a circular library or a concatemeric product generated by rolling circle amplification (RCA) (see question How does the rolling circle amplification (RCA) work in Bridge Capture™ and Nicking Loop™?). The resulting linear libraries typically range in size from 250 to 350 base pairs.

There are multiple optional outputs for Bridge Capture™ and Nicking Loop™. The key output of both technologies is the circular library (see question How is the Nicking Loop™ conversion & indexing done?), from which the other outputs are derived if needed. The circular library can be achieved in multiple ways with Bridge Capture™ or Nicking Loop™. The circular library can be directly sequenced with a circular DNA -based sequencing technology, such as Complete Genomics’s DNBSEQ , Element’s Aviti and PacBio’s Onso. Prior to sequencing the circular library can also be selectively accessed or unwanted parts of it can be deleted and still retain the original circular library for future use due to the possibility of concurrent circular DNA amplifications. Optionally, the circular library can be processed further by amplifying it with RCA (see question How does the rolling circle amplification (RCA) work in Bridge Capture™ and Nicking Loop™?), making the newly formed ssDNA concatemer directly adaptable to long-read sequencing platforms, such as Oxford Nanopore Technologies. From the amplified ssDNA concatemer the individual monomers can be amplified with a limited cycle indexing PCR (see question How does the limited-cycle indexing PCR work in the Bridge Capture™?). The amplicons are traditional linear libraries which can be sequenced with short-read platforms like Illumina and Ion Torrent.

Yes. The Nicking Loop™ workflow has been demonstrated with Opentrons OT-2.