IMPROVEMENTS IN NUCLEIC ACID SEQUENCING

Abstract

A method of preparing templates for high throughput nucleic acid sequencing, comprising: a) providing a solid support, wherein said solid support comprises a plurality of nucleic acid primers immobilised adaptor nucleic acid sequences which hybridise to one or more of the nucleic acid primers, and the library preparation having a nucleic acid concentration of 400 pM or less; c) allowing single stranded fragments of template nucleic acid to bind to said nucleic acid primers, thereby immobilising said single stranded fragments on said solid support; and d) repeating steps b) and c) at least three further times, i.e. multiple loadings or rounds of template hybridization to improve occupation rates, e.g., of nanowells on a flow cell.

Claims

1. A method of preparing a template for a nucleic acid sequencing reaction, the method comprising: a) providing a solid support, wherein said solid support comprises a plurality of nucleic acid primers immobilised thereon; b) contacting a nucleic acid library preparation with the solid support, the library preparation comprising a plurality of single stranded fragments of template nucleic acid to be sequenced, the template nucleic acid fragments further comprising one or more adaptor nucleic acid sequences which hybridise to one or more of the nucleic acid primers, and the library preparation having a nucleic acid concentration of 400 pM or less; c) allowing single stranded fragments of template nucleic acid to bind to said nucleic acid primers, thereby immobilising said single stranded fragments on said solid support; and d) repeating steps b) and c) at least three further times; to thereby provide a solid support having single stranded fragments of template nucleic acid immobilised thereon.

2. (canceled)

3. The method of claim 1, wherein an effective nucleic acid concentration calculated as number of cycles of step (c) times library nucleic acid concentration is at least 800 pM.

4. The method of claim 1, wherein the library preparation has a nucleic acid concentration of 250 pM or less.

5. The method of claim 1, wherein repetition of the contacting step takes place with the same library preparation as used in step (b).

6. The method of claim 1, comprising denaturing a library preparation comprising double stranded fragments of template nucleic acid to be sequenced, to obtain the single stranded fragments of template nucleic acid to be sequenced used in step (b).

7. The method of claim 1, further comprising amplifying the immobilised single stranded fragments of nucleic acid, to thereby generate multiple copies of the fragments.

8. The method of claim 1, wherein the solid support is glass.

9. The method of claim 1, wherein said solid support is on a flowcell, wherein said solid support has a plurality of nanowells formed thereon, each nanowell comprising a plurality of nucleic acid primers immobilised on the solid support, and the nanowells being formed in a patterned array.

10. The method of claim 9, wherein a pitch of the nanowell array is 500 nm or less.

11. The method of claim 10, wherein the pitch of the nanowell array is 350 nm or less.

12. The method of claim 1, dependent on claim 1, wherein the solid support is a microbead.

13. The method of claim 1, further comprising sequencing the immobilised single stranded fragments of nucleic acid.

14. A method of preparing a template for a nucleic acid sequencing reaction, the method comprising: a) providing a flow cell comprising a solid support having a plurality of nanowells formed thereon, each nanowell comprising a plurality of nucleic acid primers immobilised on the solid support, and the nanowells being formed in a patterned array having a pitch of 500 nm or less; b) contacting a nucleic acid library preparation with the flow cell, the library preparation comprising a plurality of single stranded fragments of template nucleic acid to be sequenced, the template nucleic acid fragments further comprising one or more adaptor nucleic acid sequences which hybridise to one or more of the nucleic acid primers, and the library preparation having a nucleic acid concentration of 400 pM or less; c) allowing single stranded fragments of template nucleic acid to bind to said nucleic acid primers, thereby immobilising said single stranded fragments in the nanowells; and d) repeating steps b) and c) at least three further times; to thereby provide a flow cell having single stranded fragments of template nucleic acid immobilised in nanowells.

15. The method of claim 14, wherein an effective nucleic acid concentration calculated as number of cycles of step (c) times library nucleic acid concentration is at least 800 pM.

16. The method of claim 14, wherein the library preparation has a nucleic acid concentration of 250 pM or less.

17. The method of claim 14, wherein repetition of the contacting step takes place with the same library preparation as used in step (b).

18. The method of claim 14, comprising denaturing a library preparation comprising double stranded fragments of template nucleic acid to be sequenced, to obtain the single stranded fragments of template nucleic acid to be sequenced used in step (b).

19. The method of claim 14, further comprising amplifying the immobilised single stranded fragments of nucleic acid, to thereby generate multiple copies of the fragments.

20. The method of claim 14, wherein the pitch of the nanowell array is 350 nm or less.

21. The method of claim 14, further comprising sequencing the immobilised single stranded fragments of nucleic acid.

Description

[0073] These and other aspects of the invention will now be described with reference to the accompanying Figures, in which:

[0074] FIG. 1 shows a schematic representation of an illustrative flow cell which may be used with certain methods described herein.

[0075] FIG. 2 illustrates the concept of the multi hybridisation workflow.

[0076] FIG. 3 illustrates results obtained using from 1 to 10 pushes (hybridization cycles) for various metrics.

[0077] FIG. 4 compares library seeding efficiency with two different multi hybridization workflows.

[0078] FIG. 5 gives results from a 4-push workflow using a 200 pM library concentration.

[0079] Referring to FIG. 1, this shows a schematic representation of an illustrative flow cell which may be used with certain methods described herein. The flow cell is formed in three layers. The bottom layer 1 is formed of borosilicate glass at a depth of 1000 μm. An etched silicon channel layer (100 μm depth) is placed on top to define 8 separate reaction channels. Top layer 3 (300 μm depth) includes two separate series of 8 holes 4 and 4′ in register with the channels of the etched silicon channel layer in order to provide fluid communication with the contents of the channels when the flow cell is assembled in use. The borosilicate glass of the bottom layer 1 is etched with nanowells set out in a patterned array having a 350 nm pitch, and arranged in line with the reaction channels. The interstitial regions—that is, between the channels—are not etched, and do not include nanowells. In use, primers for nucleic acid fragment template capture are bound to the nanowells.

[0080] To load the flow cell with a prepared library for sequencing, the following protocols may be used. An initial library of double stranded DNA including appropriate adapters for the sequencing technique to be used may be prepared in any suitable manner known in the art. For example, library preparation kits may be purchased from Illumina, Inc (San Diego, USA) to prepare suitable libraries. The examples described herein were prepared using a TruSeq Human Nano 450 preparation kit.

[0081] The below workflow models a 4 push multi hybridization recipe. The total number of hybridization events can be varied up or down depending on the necessary use case and the desired final effective concentration.

[0082] Library Denaturation and Dilution: [0083] 1. 20 ul of double stranded DNA library is diluted to the appropriate concentration with H.sub.2O or RSB Buffer (available in the TruSeq Human Nano 450 preparation kit). In order to take advantage of the multi hybridization workflow, this working concentration is 4 times lower than the concentration necessary for a standard single event hybridization protocol. [0084] 2. Mix the library 1:1 with LDR denaturation reagent (100% formamide). [0085] 3. Heat to 65° C. and incubate for 8 minutes to denature the double stranded DNA template. [0086] 4. Add 160 ul of HT1 (5×SSC+0.1% Tween20) to dilute the denatured library to the final intended working concentration. [0087] 5. Proceed to Template Hybridization

[0088] Template Hybridization with Multi Hybridization workflow, using an Illumina NovaSeq6000 system: [0089] 1. Prime/wet the cartridge lines and flow cell with BB6 buffer. [0090] 2. Heat the flow cell to 40° C. [0091] 3. Pump denatured and diluted template to the flow cell with an initial flush factor large enough to fully cover the flow cell without dilution from the upstream BB6 buffer. [0092] 4. Incubate for 5 minutes. [0093] 5. After incubating, pull an additional flow cell volume of denatured and diluted template to the flow cell and incubate for 5 minutes. [0094] 6. Repeat step 5 two additional times for a total of 4 total hybridization events. [0095] 7. Proceed to cluster generation.

[0096] FIG. 2 illustrates the concept of the multi hybridisation workflow. A typical single push workflow is shown in the top line, where template hybridization takes place once, followed by cluster generation. If the template is loaded at 800 pM concentration, the effective concentration remains 800 pM. The second and third lines set out alternate ways to achieve the same concentration; using two hybridization cycles of 400 pM (second line), or four hybridization cycles at 200 pM (third line). It can be seen that multiple rounds of template hybridization/capture at low DNA concentration increase effective DNA concentration bringing them into the necessary range. This in turn significantly lowers library prep concentration requirements and reduces template waste due to system dead volumes, fluidic lines, etc, and will potentially enable run requeues of low yield library preparations.

[0097] Using the above methodology, an initial 2 nM library was prepared and diluted to 200 pM concentration.

TABLE-US-00001 Volume Concentration (ul) (nM) Library  20 2 LDR  20 n.a. HT1 160 n.a. Final 200 0.2

[0098] Typical library yields for different applications are shown below:

TABLE-US-00002 Representative Library Applications LP assay yield (nM) Gene Expression, Whole Transcriptome TruSeq ~20 nM (RNAseq) Single Cell (Gene expression) SureCell 1 nM Somatic Mutations (Amplicon/ AmpliSeq 2 nM enrichment) TST170 ~10 nM Whole Exome Titanium 12 nM Hu-WGS, PCR-free TruSeq 2 nM Nextera (Viper) Hu-WGS, PCR TruSeq Nano 10 nM Nextera Flex

[0099] FIG. 3 illustrates results obtained using from 1 to 10 pushes (hybridization cycles) for various metrics, with each workflow being designed to provide 800 pM effective concentration. The metrics measured included % cluster formation, % occupied nanowells, % remaining duplicates, and % usable yield. It can be seen that, regardless of the number of pushes, the metrics remain within a small band, and multiple pushes up to 10 provide similar metrics as a single push of higher concentration. The optimum for usable yield and cluster formation lies within the 4-6 push range. The flow cell used had a 350 nm nanowell pitch.

[0100] FIG. 4 compares library seeding efficiency with two different multi hybridization workflows (2-push vs 4-push). An initial known quantity of library is hybridized to the flow cell in one or multiple rounds, then the template that was hybridized is eluted and qPCR quantified. The initial input and uncaptured template fraction can be quantified with hybridized fraction. The graph shows that 2-push and 4-push provide similar numbers of molecules of ssDNA per nanowell, increasing as total DNA exposure increases up to 700 pM. As long as the final DNA exposure is maintained, total push number can be varied to give similar results. The 2-push protocol utilizes 2× the DNA concentration relative to the 4-push protocol, but maintains similar final molecules per nanowell.

[0101] FIG. 5 gives results from a 4-push workflow using a 200 pM library concentration (800 pM effective concentration), on a 350 nm pitch flow cell using the library prepared with a TruSeq Human Nano 450 kit. As total DNA concentration increases to 800 pM, it can be seen that the % occupied nanowells increases, and the % duplication decreases. It can be seen that as concentration increases to 800 pM, % Pass Filter remains consistent showing that the maintaining lower concentrations over multiple pushes results in optimal seeding.

[0102] Hence the multiple hybridization workflow appears to be an effective protocol for efficient loading of high density nanowell flow cells without unduly increasing duplicated clusters, and demonstrates that it is possible to use a sequencing flow cell as a DNA capture device in order to increase the effective concentration of a sample to be sequenced, particularly when the sample preparation is of relatively low concentration yield. The preferred four-fold hybridization protocol reduces library input concentration requirements by four times, and potentially enables run requeues of low yield library preps.