TITLE: Direct oligonucleotide sequencing with nanopores
AUTHORS: Sachin Chalapati, Conor Crosbie, dixita limbachiya, Nimesh Pinnamaneni

[DESCRIPTION]
Third-generation DNA sequencing has enabled users to sequence long, unamplified DNA fragments with minimal steps. Direct sequencing of ssDNA or RNA gives valuable insights like base-level modifications, an estimate of phosphoramidite synthesis yield and analysis of strand quality, without the need to add the complimentary strand. Direct sequencing of single-stranded nucleic acid species is challenging as they are non-compatible to the double-stranded sequencing adapters used by the manufacturers. The MinION platform from Oxford Nanopore Technologies performs sequencing by passing single-strands of DNA through a layer of biological nanopore sensors, although the sequencing is performed on single-strands of DNA, the recommended template by the instrument manufacturer is still double-stranded. We have identified that the MinION platform from Oxford Nanopore can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing a single annealing step before library preparation. Short 5’ phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores. Adapter sequences were designed to bind to the 5’ end of the oligos and to leave a 3’ adenosine overhang after binding to their target. The 3’ adenosine overhang of the adapter and the terminal phosphate makes the 5’ end of the oligo analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal sequences were phosphorylated and sequenced using this method and ~90% of these sequences were recovered with high accuracy using BLAST. In the nanopore raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single raw sequence file due to segmentation faults in the software. This direct oligonucleotide sequencing method enables novel applications in DNA data storage systems where short oligonucleotides function as the primary information carriers.

[STEPS]
SECTION: Normalization
1. Normalize 3xr6 dried oligo-pool to 0.25 uM with TE buffer and verified with Qubit 4 using Qubit ssDNA Assay Kit
SECTION: Normalization
2. INS3 and EINS3 are diluted from their stock concentrations to 0.5 uM with TE buffer and verified with Qubit 4 using Qubit ssDNA Assay Kit
SECTION: Normalization
3. INS3 RC, EINS3 RC and ArcFP oligos are normalized by the supplier at 100 uM concentration and are diluted to 1 uM concentration.
SECTION: Phosphorylation of 3xr6
4. Mix the following reagents into a PCR tube.

 


SECTION: Phosphorylation of 3xr6
5. Incubate at 37 °C for 30 min 

SECTION: Phosphorylation of 3xr6
6. Heat inactivation at 65 °C for20 min 

SECTION: Phosphorylation of 3xr6
7. The mixture is washed using Monarch spin columns with the standard oligonucleotide cleanup protocol. The purified DNA is eluted into 9 ul of IDTE instead of the given Elution buffer.
SECTION: Library preparation of INS3
8. Create a triplicate of the following reaction mix:


 

SECTION: Library preparation of INS3
9. The reaction mixtures are heated to 94 °C  for 2 min  and gradually cooled to room temperature for annealing.

SECTION: Library preparation of INS3
10. 5 µL of AMX from the ligation sequencing kit is added to each of the tubes.
5 µL  of Blunt/TA master mix is added to each of the tubes.

SECTION: Library preparation of INS3
11. Tubes are incubated at room temperature for 10 min 

SECTION: Library preparation of INS3
12. Three new MinION flow cells are used to sequence each reaction mix of the triplicate and the sequencing is performed with the standard parameters for 20 min 

SECTION: Library preparation of EINS3
13. Create a triplicate of the following reaction mix:


 


SECTION: Library preparation of EINS3
14. The reaction mixtures are heated to 94 °C  for 2 min  and gradually cooled to room temperature for annealing.

SECTION: Library preparation of EINS3
15. 5 µL of AMX from the ligation sequencing kit is added to each of the tubes.
5 µL  of Blunt/TA master mix is added to each of the tubes.

SECTION: Library preparation of EINS3
16. Tubes are incubated at room temperature for 10 min .

SECTION: Library preparation of EINS3
17. Three MinION flow cells from the INS3 run are used to sequence each reaction mix of the triplicate and the sequencing is performed with the standard parameters for 20 min .
SECTION: Library preparation of 3XR6
18. Elution of the phosphorylation step is split 3-ways for triplicate sequencing runs. A triplicate of the following reactions is performed:

 


SECTION: Library preparation of 3XR6
19. The reaction mixtures are heated to 94 °C  for 2 min  and gradually cooled to room temperature for annealing.

SECTION: Library preparation of 3XR6
20. 5 µL of AMX from the ligation sequencing kit is added to each of the tubes.
5 µL  of Blunt/TA master mix is added to each of the tubes.

SECTION: Library preparation of 3XR6
21. Tubes are incubated at room temperature for 10 min .

SECTION: Library preparation of 3XR6
22. Three new MinION flow cells are used to sequence each reaction mix of the triplicate and the sequencing is performed with the standard parameters for 240 min .