IMPROVED NGS WORKFLOW

Abstract

The present invention relates to improved semi-automated methods that permit the extraction of nucleic acids from samples, preparation of PCR and post-PCR preparation steps of DNA- libraries for next-generation sequencings methods that can be conducted. The methods and additional aspects relating to such methods are less laborious, safe costs, reagents and are less prone to contamination than comparable methods that are not automated.

Claims

1. A method of preparing a DNA library comprising the steps: a) extracting nucleic acids from a sample, b) exposing the extracted nucleic acids to a mixture comprising UDG, a DNA polymerase, and dUTP, c) incubating the mixture to decontaminate the mixture from one or more carry over amplification products derived from a prior amplification reactions, d) performing an amplification reaction in the presence of dUTP, wherein the steps of b), c) and d) are performed in the same reaction mixture.

2. The method according to claim 1, wherein the DNA polymerase is a Thermus aquaticus (Taq) DNA polymerase, or a functional derivative thereof, wherein the functional derivative of Taq polymerase has at least 80% of the DNA polymerization activity of Taq polymerase.

3. The method according to claim 1, wherein the extracted nucleic acids are fragmented prior to step b).

4. The method according to claim 1, wherein the DNA library is subsequently used in a next generation sequencing reaction.

5. A reagent composition comprising an enzyme mix comprising a UDG, and a DNA polymerase.

6. The reagent composition according to claim 5 further comprising dUTP.

7. The reagent composition comprising Taq DNA Polymerase or a functional derivative thereof.

8. The reagent composition comprising reagents for reverse transcription and/or PCR.

9. A method of decontaminating a reaction mixture for the amplification of nucleic acid templates comprising: exposing said nucleic acid templates to a DNA polymerase, a UDG enzyme, and dUTP, and reagents for DNA polymerization.

10. The method according to claim 9, wherein the UDG enzyme is inactivated after a period sufficient to decontaminate the mixture from carry over amplification products derived from prior amplification reactions.

11. The method according to claim 9, wherein the DNA polymerase is a Thermus aquaticus (Taq) DNA polymerase, or a functional derivative thereof.

12. The method according to claim 9, wherein the extracted nucleic acids are fragmented prior to step b).

13. The method according to claim 9, wherein the DNA library is subsequently used in a next generation sequencing reaction.

14. A method for the preparation of a DNA library comprising the steps: a) extracting nucleic acids from a sample, b) exposing the extracted nucleic acids to a mixture comprising a DNA, c) performing an amplification reaction in the presence of dUTP, d) normalizing the obtained amplification products, wherein the normalizing the obtained amplification products comprises the following steps: (i) adding a buffer composition comprising an alkali metal salt and a solvent to the amplification mixture comprising amplification products, (ii) adding carrier particles to the amplification mixture comprising amplification products, (iii) incubating the mixture for a time sufficient for the DNA to bind to the carrier particles, (iv) washing the mixture with ethanol, (v) elution of normalized PCR products from the carrier particles.

15. The method according to claim 14, wherein the alkali metal salt is NaCl.

16. The method according to claim 14, wherein the solvent polyethylene glycol.

17. The method according to claim 14, wherein the alkali metal salt added in an amount of about 2.0 to about 5.0 M NaCl.

18. The method according to claim 14, wherein the solvent is PEG 8000.

19. The method of claim 1, wherein the mixture of step b) further comprises a reverse transcriptase.

20. The method according to claim 2, wherein the functional derivative of Taq polymerase has at least 90% of the DNA polymerization activity of Taq polymerase.

21. The method according to claim 2, wherein the functional derivative of Taq polymerase has at least 100% of the DNA polymerization activity of Taq polymerase.

22. The reagent composition of claim 5, further comprising a reverse transcriptase.

23. The method of claim 9, wherein the nucleic acid templates are further exposed to reverse transcriptase and reagents for reverse transcriptase

24. The method of claim 14, wherein the mixture of step b) further comprises a reverse transcriptase.

25. The method according to claim 14, wherein the alkali metal salt added in an amount of about 2.0 to about 2.5 M NaCl.

Description

EMBODIMENTS OF THE PRESENT INVENTION

[0041] The present invention relates, amongst others, to unique semi-automated methods for the isolation of nucleic acids from samples, set-up of (RT-)PCR reaction, (RT-)PCR-based nucleic acid amplification, post-PCR normalization and clean up of amplification products, fragmentation of PCR amplification products, ligation with adaptors characterized by the following steps set out in (A) and (B):

[0042] Method (A) [0043] (a) Extraction of nucleic acids from a sample; [0044] (b) Optionally addition of Uracil-DNA-glycosylase (UDG) to the (RT-) PCR mixture before conducting (RT-) PCR reaction to digest cross and carryover contamination from prior amplification reactions; [0045] (c) (RT-)PCR, depending on type of isolated nucleic acids, i.e. RNA or DNA, using nucleotide triphosphate building blocks (i.e. individual nucleotides) comprising A, T, C, G, optionally also comprising Uracil; [0046] (d) Normalization of nucleic acids obtained in RT-PCR (using carrier structures, e.g. paramagnetic microbeads (e.g. AxyPrep Mag PCR Normalizer, Axygen) for normalization, wherein said beads bind nucleotide sequences of a desired sequence length) comprising binding RT-PCR mixture subsequent to PCR to said beads, thoroughly washing the microbeads subsequent to binding of PCR-product, elution of PCR amplification products from microbeads; [0047] (e) Fragmentation (Shearing) eluted PCR amplification products obtained in step (d); [0048] (f) Binding the product of step (e) to carrier structures, e.g. microbeads, followed by washing and elution of the bound nucleic acids; [0049] (g) Ligation of adaptor sequences (comprising barcode sequences allowing attribution of nucleic acids to specific sample (e.g. clinical sample and patient) to the product obtained in step (f); [0050] (h) Cleaning up the product obtained in step (g) using carrier structures, e.g. microbeads used in previous steps (d) and/or (f); [0051] (i) Subjecting the product obtained in step (h) to sequencing reaction (e.g. using Ion PGM System), and [0052] (j) Analysis of the results of the sequencing reaction obtained in step (i).

[0053] Method (B) [0054] (a) Extraction of nucleic acids from a sample; [0055] (b) Optionally addition of Uracil-DNA-glycosylase (UDG) to the (RT-) PCR mixture before conducting (RT-) PCR reaction to digest cross and carryover contamination from prior amplification reactions; [0056] (b) RT-PCR, depending on type of isolated nucleic acids, i.e. RNA or DNA, using nucleotide triphosphate building blocks (i.e. individual nucleotides) comprising A, T, C, G, optionally also comprising Uracil; [0057] (d) Partial digestion of primers (e.g. using FuPa reagent of Life Technologies); [0058] (e) Ligation of adaptor sequences (comprising barcode) to the product obtained in step (d); [0059] (f) Normalization of nucleic acids obtained in RT-PCR (using carrier structures, e.g. paramagnetic microbeads (e.g. AxyPrep Mag PCR Normalizer, Axygen) for normalization, wherein said beads bind nucleotide sequences of a desired sequence length) comprising binding RT-PCR mixture subsequent to PCR to said beads, thoroughly washing the microbeads subsequent to binding of PCR-product, elution of PCR amplification products from microbeads; [0060] (g) Clean up product obtained in step (g) using carrier structures, e.g. microbeads used in previous steps (d) and/or (f); [0061] (i) Subject product obtained in step (h) to sequencing reaction (e.g. using Ion PGM System; Ion Torrent), and [0062] (j) Analysis of the results of the sequencing reaction obtained in step (i).

[0063] The uniqueness of the above workflow methods allows reducing the amount of time required in the process from the extraction of the nucleic acids for analysis and the final NGS reaction, which is followed by analysis of the results. The use of UDG largely reduces the risk of contamination in automated systems for nucleic acid extraction, PCR set-up, post-PCR purification steps, library preparation and NGS. Automation of these steps using the above methods reduces the time and costs required in particular for diagnostic applications.

[0064] Surprisingly, it was noticed that an innovative alternative method described herein can be used in preparation of next-generation sequencing libraries. The inventive methods can be used in the preparation of different types of NGS-libraries, e.g. for Illumina sequencing or for Ion Torrent sequencing. This method can be incorporated into the NGS workflow set out above.

[0065] Usually, NGS libraries are prepared using commercially available kits including buffers that are suitable for such purpose. These buffers are specifically optimized for the robust, high-fidelity amplification of NGS-libraries, regardless of the GC-content. As automated open systems for the preparation of NGS libraries can be susceptible to the high risk for the carry-over of contamination of clinical samples by PCR amplicons from previous runs, it is an objective to reduce said danger. To prevent carry-over contamination, dUTP is added to (RT-) PCR master mixes. Uracil dehydrogenase (UDG)-treatment of PCR master mixes removes contaminant amplicons from previous runs and that may accidentally have been carried over into subsequent reaction mixtures. Uracil dehydrogenase (UDG) is an enzyme that removes uracil from DNA by hydrolysis of the N-glycosylic bond between the deoxyribose and the base leaving an apurinic or apyrimidinic site (AP site).

[0066] However, buffers for NGS-library preparation (e.g. SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity) generally are not suitable for the incorporation of dUTP during amplification reactions. It was surprisingly noticed that specific high fidelity enzymes specifically developed for NGS-library preparation can be replaced by conventional Taq Polymerase, which are non-high fidelity enzymes. Conventional (RT-) PCR buffer (e.g. buffers containing 100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl.sub.2) can be used. This modification in the protocol for preparation of NGS-libraries allows incorporation of dUTP during amplification.

[0067] Accordingly, in one aspect the present invention relates to methods of preparing NGS libraries comprising incorporation of dUTP during amplification without using specialized high fidelity PCR buffers, but wherein essentially any DNA polymerase (e.g. Taq Polymerase) that is suitable for PCR is used. This rather simple exchange of buffers and enzymes allow the introduction of dUTP and subsequent treatment with UDG to prevent carry-over of contaminants.

[0068] Further, the invention relates to a method for elimination of carry-over contamination, i.e. for the decontamination of reagent mixtures comprising extracted nucleic acids that should be analysed and potentially contaminating DNA derived from previous (RT-)PCR reactions, in nucleic acid amplification reactions for the preparation of a next generation sequencing library using wild-type (recombinant or native) Taq polymerase for the incorporation of dUTP, comprising the steps of: [0069] a) fragmenting nucleic acids obtained from a sample, [0070] b) adding a degrading enzyme suitable to degrade any contaminating nucleic acid amplificates present in the amplification reaction mixture; [0071] c) amplifying a nucleic acid template in order to provide a first nucleic acid amplificate in a first nucleic acid amplification reaction in the presence of dUTP; and [0072] d) inactivating said degrading enzyme.

[0073] In a preferred embodiment of the above method for elimination of carry-over contamination in nucleic acid amplification reactions for the preparation of a next generation sequencing library using wild-type (recombinant or native) Taq polymerase or a derivative thereof for the incorporation of dUTP, the degrading enzyme is UDG. The UDG treatment usually takes several minutes, e.g. up to 10 minutes, preferably up to 5 minutes. Subsequently, the enzyme is deactivated, e.g. by exposure to temperatures of about 50 C. for about 5 minutes.

[0074] In another preferred embodiment of the above method for elimination of carry-over contamination in nucleic acid amplification reactions for the preparation of a next generation sequencing library using wild-type Taq polymerase for the incorporation of dUTP, the degrading enzyme is UDG the Taq Polymerase is recombinant or native polymerase.

[0075] In preferred embodiment of the above method for elimination of carry-over contamination in nucleic acid amplification reactions for the preparation of a next generation sequencing library using wild-type (recombinant or native) Taq polymerase for the incorporation of dUTP, the degrading enzyme is UDG, and the UDG-treated library is subjected to further steps in the next generation sequencing method, comprising: [0076] a) fragmenting nucleic acids obtained from a sample, [0077] b) adding a degrading enzyme suitable to degrade any contaminating nucleic acid amplificates present in the amplification reaction mixture; [0078] c) amplifying a nucleic acid template in order to provide a first nucleic acid amplificate in a first nucleic acid amplification reaction in the presence of dUTP; and [0079] d) inactivating said degrading enzyme.

[0080] Preferred embodiments of the present methods for the generation of DNA libraries, or the decontamination of reaction mixtures in the process of the above DNA library preparation are also depicted in the claims.

[0081] Preferred embodiments of methods (A) and (B) above relate to in vitro diagnostic applications, e.g. in companion diagnostics where knowledge about the sequence of a target nucleic acid (for example, an oncogene or a nucleic acid derived from a pathogen like HCV, HIV, or the like) present in a clinical sample helps the physician to select the most promising treatment for a patient, because modifications in some oncogenes confer resistance to certain drugs.

[0082] In a preferred embodiment of method (A), the sample is a fresh sample obtained, e.g. from a patient, preferably a human patient. The sample material may be, for example, blood, plasma, a subpopulation of blood cells, e.g. T-cells, cerebrospinal fluid, sputum, stool, and the like.

[0083] In a preferred embodiment of method (A), the sample is plasma in order to isolate nucleic acid material found therein, e.g. viral, bacterial, fungal, or parasite-derived nucleic acids or material containing such nucleic acids, e.g. virions, bacteria, and the like.

[0084] In a preferred embodiment of method (A) the sample material is plasma and the nucleic acid material is derived from a virus, e.g. HCV, HIV, etc. When HCV is targeted, the region of interest is preferably the NS5B gene region, which is well-suited to identify 6 major HCV genotypes and a large number of subtypes. The target region in of the HCV genome is preferably extending from nucleotide 8616 to nucleotide 9298, but the region may be slightly longer or shorter as long as the identification of 6 HCV genotypes is possible. Preferred primers bind to nucleotides 8616-8638, 8614-8635, 9276-9298 and 9171-9191 of the HCV genome. The primers may comprise natural or modified nucleotide building blocks as known in the art.

[0085] In a preferred embodiment of method (B), the sample is a fresh sample obtained, e.g. from a patient, preferably a human patient. The sample material may, for example, be blood, plasma, a subpopulation of blood cells, e.g. T-cells, cerebrospinal fluid, sputum, stool, and so forth. In another preferred embodiment of method (B), the sample is not a fresh sample, but a sample that has been treated after obtaining the same, e.g. using formalin-fixation and/or paraffin-embedding (FFPE samples are preferred samples for analysis of various oncogenes).

[0086] In a preferred embodiment of method (B), the sample material is an FFPE-sample derived from a human patient, e.g. a sample from any tissue that may be formalin-fixed and/or paraffin-embedded, e.g. a sample derived from skin, breast tissue, colon, lung, liver, muscle, etc. In a very preferred embodiment, the sample is skin sample for analysis of genes involved in melanoma formation. Preferred genes targeted in this context comprise at least one or more of the following group of genes: NRAS, AKT3, MAP2K1, GNAT 1, ERBB4, PIK3CA, FGFR3, KIT, BRAF, CDKN2A, and GNAQ. These genes are known to be involved in the development of melanoma and may contain different point mutations at different sites of the respective genes. The analysis of specific mutations allows the treating physician to choose a suitable therapy as some mutations are known to confer drug resistance, whereas others are drugable (sensitive to drugs).

[0087] Another aspect of the present invention is the provision of new FFPE cell lines that may serve as control material for nucleic acid extraction from FFPE tissue. These cell lines may carry genetic information that corresponds to the targeted sequence, e.g. genetic material that was previously introduced via transformation or using other methods. Alternatively, these genes may not have been genetically modified, e.g. when the cells already carry target genes of interest (for example oncogenes) or when the target gene should be different from the gene targeted in the actual assay. For example, when the assay targets mutations of one or more oncogenes in clinical sample, the gene targeted in the FFPE cell lines may be a house-keeping gene, or a non-mutated wildtype gene. The cell lines provide a source of quantifiable amounts of target nucleic acid, since the amount of FFPE cell line material may be selected to match the requirements of individual assays. The inventive cell lines may be provided as a part of a kit for any given assay. Said kit may further comprise additional chemical reagents suitable for the extraction, purification, amplification or other manipulation of nucleic acids, e.g. primers, buffers, enzymes, and the like.

[0088] Another aspect provided herein is a method for the normalization of DNA libraries. In further embodiments, these DNA libraries are used for subsequent NGS involving the use of carrier particles such as magnetic microbeads.

[0089] In prior methods, the normalization of DNA libraries required the quantification and/or size selection of fragmented DNA amplification products obtained in (RT-) PCR reactions before ligation of adapters. It was surprisingly found out that the library preparation involving the use of microbeads does not require size selection and/or prior quantification, preferred microbeads are those provided by Axygen (AxyPrep MAG-PCR-CL-5Kit) or similar products. The use of these microbeads eliminates also shorter fragments still present after nucleic acid amplification and/or ligation of adapters to the amplification products.

[0090] Furthermore it was surprisingly found out that the PCR amplification of thus generated DNA libraries is not necessary, unlike in prior art methods where the library comprising adapters subsequent to ligation was amplified again.

[0091] Depending on the quantity of beads and the incubation time of said beads with the DNA library, the quantity of bound DNA can be defined, since the beads are saturated with nucleic acids over time.

[0092] The inventive automated nucleic acid extraction, amplification and library preparation method (e.g. using the Sentosa SX101 platform of Vela Diagnostic) allows reducing time, amount of reagents and costs in general and avoids the risk associated with manual preparation of DNA libraries for NGS.

[0093] The present invention also contemplates a kit for the preparation of generic libraries.

[0094] Still further, the present invention provides a simplified and improved library preparation protocol. As mentioned above, normalizing magnetic beads for the preparation of DNA libraries that are used the subsequent NGS protocol are very important in order to obtain correct amounts of nucleic acids for further analysis. To this end, DNA binding beads with limited binding surface can be used after (RT-)PCR can be used for normalization of the amplified nucleic acids.

[0095] Further, to obtain a pre-defined amount of DNA for the following next generation sequencing steps, prior art methods essentially required the following steps: [0096] 1) (RT-)PCR [0097] 2) Clean-up of PCR products using magnetic beads and clean-up buffer [0098] 3) Washing the beads (e.g. with ethanol) [0099] 4) Elution of PCR product bound to magnetic beads [0100] 5) Normalization of PCR products using normalization magnetic beads and normalization buffer [0101] 6) Washing the beads (e.g. with ethanol) [0102] 7) Elution of normalized PCR product.

[0103] Normalization magnetic beads (Definition) are very sensitive to RT-PCR buffer, presumably because dTT in one-step RT-PCR buffers inhibit the binding of amplified DNA products to normalization beads. It was previously necessary in prior art methods to perform the above steps 2) to 4), which remove reagents present in RT-PCR mixture after amplification was carried out.

[0104] Surprisingly, the present inventors found out that tedious, time-consuming and costly steps 2) to 4) can be omitted when the (RT-)PCR products are exposed to a new inventive composition comprising for normalization beads for NGS library preparation comprising a solvent, e.g. polyethylene glycol and an alkali metal salt, e.g. NaCl, MgCl, or the like. In some embodiments, the composition comprises, e.g. about 2.0 to about 5.0 M NaCl, e.g. 2.0 M to about 4.0 M NaCl, preferably 2.5 M to about 3.5 M NaCl, very preferably about 2.5 to about 3.0 M NaCl, and most preferably the concentration of the alkali metal in the inventive buffer is 2.5 M NaCl. The inventive buffers for normalization beads for NGS library preparation further comprises about 10% to about 30% of a solvent, e.g. about 12.5% to about 25%, or 15.0% to about 25%, or 17.5% to about 22.5%, preferably about 20% of a solvent. The solvent is preferably a polyethylenglycol, e.g. high molecular weight PEG such as Polyethylenglycol (PEG) 8000. It is possible also to replace NaCl by other alkali metal salts such as Mg, K, etc. In a very preferred embodiment the inventive buffers for normalization beads for NGS library preparation comprises about 2.5 M NaCl and 20% Polyethylenglycol (PEG) 8000.

[0105] In inventive methods for the preparation of NGS libraries and the improved NGS workflow, the above-described buffer is added directly to the obtained RT-PCR amplification mixture containing the amplified nucleic acids. The inventive buffer is preferably added in ratio of 2:1 to 1:2 to the amplification mixture, and most preferably the inventive buffers are added in an about equal amount (e.g. 1:1) to the PCR amplification mixture. In a very preferred embodiment the inventive buffers for normalization beads for NGS library preparation comprises about 2.5 M NaCl and 20% Polyethylenglycol (PEG) 8000 are added in a ratio of 1:1 to the PCR amplification mixture.

[0106] The time and steps for the preparation of libraries for NGS can thus be strongly reduced. Further, the buffer added to the (RT-)PCR products is quite cheap, in particular it is much cheaper than the clean-up beads and the clean-up buffer.

[0107] The examples set out below serve only as examples and should by no means be construed as limiting the scope of the present invention.

EXAMPLES

Example 1

Preparation of an HCV library for NGS using Vela Diagnostic's automated Platform Sentosa SX101

[0108] 1. RT-PCR [0109] HCV viral RNA is isolated from human plasma and cDNA synthesized. Here, this step is performed using the automated platform Sentosa SX 101. [0110] Before RT-PCR is conducted, Uracil-DNA-glycosylase (UDG) is added to the RT-PCR mix to eliminate potential contaminants derived from prior assays. Perform amplicon-carry over contaminant digestion with UDG for 4 min at 25 C. before amplification.

[0111] 2. Normalization after RT-PCR

[0112] Reference is made to a working platform depicted in FIG. 1.

[0113] Prepare wells of Reagent 96-well plate (FIG. 1, position C1):

TABLE-US-00001 Aliquot into the Reagent Aliquot into Library Prep 96-well Plate Reagent holder B4: 75 L Shearase buffer 1A: 220 L Normalizer Beads (Axygen) (Life technologies) 1B: 500 L Mineral Oil (Sigma) C4: 50 L Shearase enzyme 1D: Empty Tube D4: 50 L Shearase enzyme 2A: 1500 L PCR clean-up buffer B6: 30 L dNTP mix (Vela Diagnostics, Inc.) C6: 100 L 10X ligase buffer: 2B: 15,000 L Clean-up beads (Axygen) D6: 50 L DNA ligase 2C and 2D: Normalizer Binding buffer E6: 30 L Nick repair (Axygen) polymerase (Enzymatics) C8: P1 + barcode 12 mix 3A: 1500 L Normalizer Elution buffer (Axygen) 3B and 3C: 1600 L Nuclease free water (BST) [0114] Set temperature of the Reagent 96-well plate (TEMP2 in FIGS. 1) to 15 C.; [0115] Pool 25 L of every PCR product (in the total of 4) of each sample to a defined position. [0116] Mix 5 and transfer 195 L of normalization beads to 1500 L PCR clean-up buffer (Lib Prep Reagent); [0117] Mix 10 and transfer 86 L of PCR clean-up buffer (Vela Diagnostics) and Normalizer beads (Axygen). Mix (Lib Prep Reagent) to defined position of pooled PCR product and mix for 10 times. [0118] Incubate for 3 min at room temperature; [0119] Transport the PCR plate to the magnetic holder at position B5 in the platform in FIG. 1; [0120] Incubate for 2 min; [0121] Discard supernatant by pipetting 40 L three times and 50 L once; [0122] Add 100 L of 80% EtOH to selected well on the PCR plate; [0123] Transfer PCR plate to the thermomixer (TMX) and shake at 1000 rpm for 2 min; [0124] transport the PCR plate back to the magnetic holder at position B5 in FIG. 1; [0125] The temperature control of the TMX is turned on and set to 56 C.; [0126] Incubate for 2 min; [0127] Discard supernatant by pipetting 70 L three times and 40 L once; [0128] Transport the PCR plate to the thermomixer (TMX) set previously to 56 C.; [0129] Dry the plate for 2 min; [0130] Transport the PCR plate to position C1; [0131] Add 35 L of elution buffer (Lib Prep Reagent 1A to PCR Plate); [0132] Mix 5 times by pipetting; [0133] Transport the PCR plate to the thermomixer; [0134] Shake for 5 min 1400rpm at 56 C. on the thermomixer; [0135] Transport plate back to the magnetic plate (B5) and wait for 2 min;

[0136] 3. Shearing [0137] Transfer 63 uL of shear buffer (Life technologies) and 30 uL to a defined position and mix 5 times. Transfer 80 ul of the mixture to shear enzyme (Life technologies) (C4 and D4) respectively mix 20; [0138] Transfer 15 ul to defined position. Transfer 15 uL of eluted sample (from step 2) to the same defined position and mix; [0139] Transport PCR plate to the thermomixer and incubate 12 min, 38 C. for 13 minutes.

[0140] 4. PCR Beads Clean-Up [0141] Mix the PCR clean up beads and transfer 50 L of the beads from Lib Prep Reagent to defined PCR plate well; [0142] Transport the PCR plate to TMX and shake at 1200 rpm for 3 min at 26 C. [0143] Transport the PCR plate to the magnetic holder at position B5 and wait for 2 min. [0144] Discard the supernatant by pipetting 70 L and 30 uL respectively [0145] Add 100 L 80% EtOH (Lib Prep Reagent to PCR Plate); [0146] Transport PCR plate back to the TMX and shake at 1200 rpm for 3 min at 26 C.; [0147] Transport PCR plate to the magnetic holder at B5. Wait for 3 min; [0148] Discard supernatant by pipetting 70 L once and 50 L once; [0149] Dry beads by waiting for 5 min; [0150] Transport the PCR plate back to location C1; [0151] Add 28 L elution buffer (transfer elution buffer from Lib Prep Reagent to selected PCR plate well); [0152] Transport PCR plate to the TMX and shake at 1200 rpm for 2 min at 26 C.; [0153] Transport PCR plate to magnetic holder on B5 and wait for 2 min;

[0154] 5. Ligation [0155] Transfer 90 L of ligase buffer (Enzymatics), 18 uL dNTP, 36 uL T4 ligase (Enzymatics), 18 uL Manta polymerase (Enzymatics), and 108 uL of water from defined reagent plate well to another defined tube and mix by 10 times; [0156] Transfer another 15 L of the mix from Reagent plate defined well to another defined well; [0157] Subsequently, transfer 10 uL of barcode adaptor to the same defined well. [0158] Transfer 25 uL of sample eluted from step 4 to the same defined well and mix by ten times. Cover the mixture with 25 uL mineral oil. [0159] Transport PCR plate to the TMX and incubate at 26 C. for 10 min; [0160] Increase the temperature to 65 C. and incubate for another 5 min.

Example 2

AmpliSeg Library Automation

[0161] Prepare wells of Reagent 96-well plate (FIG. 1, position C1) using AmpliSeg reagents (Life technologies, Inc.):

TABLE-US-00002 Aliquot the following into the Aliquot the following into the Library Reagent 96-well Plate Prep Reagent holder (position B1) A1: Primer pool 1: 10 L 1A: Elution buffer: 100 L C1: primer pool 2: 10 L 2A: Mineral oil: 300 L A3: PCR master mix: 15 L 2B: Binding buffer: 200 L A5: FuPa: 7 L 2C: Normalization beads: 40 L A7: Ligase enzyme: 7 L 2D: Nuclease-free water: 100 L C7: Switch solution: 15 L 6A: 80% ethanol: 2 mL A9: Barcode adapter mix: 8 L

[0162] Ampliseq library automation using automated platform Sentosa SX101 (Vela Diagnostics)

[0163] 1. PCR [0164] Set temperature at position TEMP2 to 4 C. [0165] Transfer 4 L of PCR master mix from defined wells in Reagent plate to primer pools in other selected well; [0166] Mix by pipetting 10; [0167] Transfer 74 of PCR mix from selected Reagent 96-well Plate wells to selected PCR 96-well Plate wells, respectively; [0168] Transfer 3 L of gDNA samples from defined Elution Plate well to selected PCR 96-well Plate wells, respectively (Total PCR Vol.=10 L); [0169] Manually seal the PCR plate and transfer to the PCR for amplification using the following program: [0170] Step 1: 99 C. 2 min [0171] Step 2: 99 C. 15 sec [0172] Step 3: 60 C. 4 min [0173] repeat step 2 (21) [0174] Hold at 10 C. [0175] After PCR, return PCR plate to C1 position on the Sentosa platform SX101 (FIG. 1); [0176] Set thermomixer temperature to 52 C.

[0177] 2. FuPa Reaction [0178] Transfer 2 L of FuPa (Life technologies) from selected Reagent 96-well Plate well to predetermined PCR 96-well Plate well. (Transferring of very small volumes of viscous reagents using an automated system); [0179] Pool 10 L of the PCR product from defined wells to well on the PCR plate that contains FuPa reagent and mix by pipetting 5; [0180] Add 40 L oil overlay to PCR Plate well of previous step. (Lib Prep reagent.fwdarw.PCR Plate); [0181] Transport the PCR Plate to the TMX and shake at 300 rpm at 52 C. for 10 min, 57 C. for 10 min, and 62 C. for 20 min; [0182] Transfer the PCR Plate back to position C1 on the SX101.

[0183] 3. Ligation Reaction [0184] Add 4 L of the Switch solution (Life technologies) from defined Reagent Plate well to other defined PCR Plate well; [0185] Transfer 2 L of ligase from defined Reagent Plate well to another defined PCR Plate well; [0186] Transfer 2 L of barcoded adapters from defined Reagent Plate well to predetermined PCR Plate well; [0187] Add 5 L of water from predetermined well containing Library Prep reagent to another predetermined PCR Plate well; [0188] Add 17 L sample and mix by pipetting 5; [0189] Transfer the entire sample from selected PCR Plate well to well which already contains the ligase and mix by pipetting 5; [0190] Add 40 L oil overlay to selected PCR Plate well B5. (Lib Prep reagent to defined PCR Plate wells); [0191] Wait for 20 min; [0192] Set thermomixer to 72 C. and wait for another 10 min; [0193] Transport the PCR Plate to the TMX and at 300 rpm at 72 C. for 10 min.

[0194] 4. Bead Normalization [0195] Mix normalization beads by pipetting for 10; [0196] Add 10 L of normalization beads in Lib Prep Reagent to 200 L of binding buffer in Lib Prep Reagent; [0197] Transport the PCR Plate from the TMX to position C1; [0198] Set the thermomixer temperature to 25 C.; [0199] Mix the beads solution in defined well for 10 before transferring 100 L of the beads solution to the desired PCR Plate well; [0200] Transfer 25 L of the sample from one selected PCR Plate well to another defined well for binding and mix by pipetting 10; [0201] Wait for 5 min; [0202] Transport the PCR Plate to the TMX; [0203] Shake at 1200 rpm for 1 min at 25 C.; [0204] Incubate for 4 min; [0205] Transport the PCR Plate to the magnetic plate holder B5 and incubate for 2 min; [0206] Discard the supernatant by pipetting 504 for 2 and 20 L for 1; [0207] Transfer 100 L of 80% EtOH to selected PCR Plate well; [0208] Transport the plate back to the TMX and shake at 1000 rpm for 1 min at 25 C.; [0209] Incubate for 1 min; [0210] Transport the PCR Plate back to the magnetic plate holder B5 and incubate for 2 min; [0211] Discard the supernatant by pipetting 50 L for 2 and 20 L for 1; [0212] Transport the plate back to the TMX and shake at 1000 rpm for 1 min at 25 C.; [0213] Incubate for 1 min; [0214] Transport the PCR Plate back to the magnetic plate holder B5 and incubate for 2 min; [0215] Discard the supernatant by pipetting 50 L for 2 and 20 L for 1; [0216] Set the TMX to 58 C.; [0217] Transport the PCR plate to the TMX to dry off the EtOH for 2 min; [0218] Transport the PCR plate back to position C1; [0219] Add 25 L of elution buffer to PCR Plate selected well; [0220] Transport the PCR plate to the TMX and shake at 1200 rpm for 2 min; [0221] Transport the PCR Plate to the magnetic plate holder B5 and incubate for 2 min; [0222] Pipette 25 L of the eluted sample from one defined PCR Plate well to another defined well.

[0223] The methods and additional aspects relating to such methods are less laborious, safe costs, reagents and are less prone to contamination than comparable methods that are not automated or require more manual steps.