-
Size selection
Pore-C DNA extracts produced by following this protocol may be expected to yield an average fragment size of <10 Kbp when sequenced natively. Moreover, short reads of non-chimeric Pore-C monomers (or singletons) will not provide any contact information. To deplete non-chimeric monomers and maximise the frequency of chimeric Pore-C polymers we recommend a SPRI size selection, following the protocol as described but using a 0.85X custom SPRI bead ratio rather than the 0.7X described. If the initial yield Pore-C DNA extract is <1 μg and insufficient for SPRI size selection see below.
-
PCR amplification
PCR amplification is not required for RE-Pore-C library prep as the Pore-C DNA extract can be sequenced natively. However, if a low yield of Pore-C DNA extract is obtained, either before or after size selection, where there is insufficient mass to proceed with a native ligation library preparation, it is possible to proceed with library preparation using a PCR-based approach (we recommend the Rapid PCR Barcoding Kit, SQK-RPB004). Libraries generated using the Rapid PCR Barcoding Kit are likely to generate more raw sequencing data compared with native (non-amplified) libraries, but tend to have an approximate number of contacts per run due to the shorter length of the amplicons generated. Therefore, the resulting data may be expected to have a higher cis:trans ratio.
-
Preparing the sequencing library
We recommend the following approaches to preparing a sequencing library, according to the initial yield of Pore-C DNA extract:
>1 μg initial extraction yield of Pore-C DNA extract
- Proceed with SPRI size selection, following the input requirement as detailed in the size selection protocol. Expect a recovery of approximately 80% relative to the input mass. Analyse ~100 ng of size-selected Pore-C DNA extract on an Agilent Bioanlyser 12,000 bp chip for average fragment size. We have found Pore-C DNA extracts obtained using either DpnII or NlaIII in situ digestion yield average fragment lengths of approximately 7 kb following SPRI size selection.
- Proceed with a Ligation Sequencing Kit. We recommend the SQK-LSK114 kit for maximum sequencing accuracy and output. Older versions of the kit: SQK-LSK110 or SQK-LSK109 can also be used. Library preparation requires 100–200 fmols of SPRI size-selected Pore-C DNA extract as input. Follow the library preparation protocol as stated, however when it comes to the combined FFPE + Ultra II DNA repair and end-prep, incubate the reaction at 20°C for 15 minutes then 65°C for 5 minutes. Do not incubate at 20°C for 5 minutes then 65°C for 5 minutes as stated in the Ligation Sequencing Kit protocol. Before loading the flow cell, please see the advice on maximising throughout below.
- Lower molar inputs may be considered, however if SPRI size selection yields <40 fmols Pore-C DNA extract, proceed with a Rapid PCR Barcoding Kit (SQK-RPB004) library preparation.
500 ng–1 μg initial extraction yield of Pore-C DNA extract
- Insufficient mass of Pore-C DNA extract to carry out a SPRI size selection.
- Proceed with a Ligation Sequencing Kit. We recommend the SQK-LSK114 kit for maximum sequencing accuracy and output. Older versions of the kit: SQK-LSK110 or SQK-LSK109 can also be used. Library preparation requires 100–200 fmols of SPRI size-selected Pore-C DNA extract as input. Follow the library preparation protocol as stated, however when it comes to the combined FFPE + Ultra II DNA repair and end-prep, incubate the reaction at 20°C for 15 minutes then 65°C for 5 minutes. Do not incubate at 20°C for 5 minutes then 65°C for 5 minutes as stated in the Ligation Sequencing Kit protocol. Before loading the flow cell, see the advice on maximising throughout below.
100 ng–500 ng initial extraction yield of Pore-C DNA extract
- Proceed with a Rapid PCR Barcoding Kit (SQK-RPB004) library preparation following input requirements as detailed in the protocol.
-
Maximising output and accuracy
Pore-C sequencing libraries made using the Ligation Sequencing Kit (SQK-LSK110 or SQK-LSK109) and sequenced on R9.4.1 flow cells may be expected to yield 1–2 Gb in 6 hours, or 4–8 Gb in 48 hours per flow cell on MinION Mk1B/GridION, as Pore-C DNA extracts are prone to blocking the pores. For this reason, we recommend initially loading flow cells with 20–40 fmols of sequencing library (double this for PromethION flow cells) and washing the flow cell using the Flow Cell Wash Kit (EXP-WSH004) to increase throughput.
Pore-C libraries made using the Ligation Sequencing Kit V14 (SQK-LSK114) and sequenced on R10.4.1 flow cells are expected to yield similar outputs per flow cell. Using this chemistry will yield greater resolution of repeat-rich sequences, which, in combination with the chromatin conformation contact mapping, would improve scaffolding of these regions in genomic assemblies (such as centromeres). Improved accuracy is beneficial to help scaffold "dark" regions of the genome such as the centromeres.
Over the course of the sequencing run, within approximately 18 hours, the number of available channels will likely decrease. When fewer than 20% of the channels remain available, we strongly recommend a flow cell wash to restore pore availability: this will require the Flow Cell Wash Kit (EXP-WSH004). Following the wash, reload the flow cell with another 20–40 fmols of sequencing library (double this for PromethION flow cells) - this will require the Sequencing Auxiliary Vials expansion (EXP-AUX001 if using SQK-LSK109, and EXP-AUX002 for SQK-LSK110 or EXP-AUX003 for SQK-LSK114). Repeating this process every time fewer than 20% of the channels remain available will optimise throughput.
The following kit requirements are based on several assumptions:
- a final library prep yield of >100 fmols
- a pore blocking profile that will require flow cell washes and re-loading of library within 18 hours or less
- a total run time of 72 hours
For a MinION Mk1B/GridION flow cell, you will need:
Kit Number of kits needed per flow cell Number of kits needed to maximise the use of one Ligation Sequencing Kit Ligation Sequencing Kit 1 1 Flow Cell Wash Kit (EXP-WSH004) 1 6 Sequencing Auxiliary Vials expansion 1 1 For a PromethION flow cell, you will need:
Kit Number of kits needed per flow cell Number of kits needed to maximise the use of one Ligation Sequencing Kit Ligation Sequencing Kit 1 1 Flow Cell Wash Kit (EXP-WSH004) 1 3 Sequencing Auxiliary Vials expansion 1 1 Note: flow cell washes are not required for sequencing libraries generated using a Rapid PCR Barcoding Kit (SQK-RPB004).