A method for determining the number of PCR amplification cycles

Abstract

A method for determining the number of PCR amplification cycles includes determining the number of PCR amplification cycles for a sample according to the following formula: Cycle number y = DV200 * (-4.5223) + lg(initial RNA input) * (-3.5) + K, where K is 26.7821 or 28.7821; the cycle number y is rounded to the nearest integer. This invention effectively avoids over-amplification, ensuring that a large number of samples remain within a reasonable library concentration range.

Description

Technical Field

[0001] This invention relates to the field of gene detection, and more specifically to a method for determining the number of PCR amplification cycles. Background Technology

[0002] With an aging and rapidly growing population, the number of cancer cases and deaths worldwide is also increasing rapidly. To maximize the benefits and minimize the toxicity of cancer treatment, targeted therapy has become one of the main approaches to cancer control, achieving impressive results for some cancers. These targets are either surface markers, such as human epithelial receptor 2 (HER2) (targeted by trastuzumab), or internal markers, such as BRAF V600E mutations (targeted by vemurafenib) and EML4-ALK fusions (targeted by crizotinib).

[0003] Fusion genes are formed by structural chromosomal rearrangements (primarily including translocations, inversions, amplifications, and deletions) or non-structural aberrations caused by cis- and trans-splicing or transcriptional readthrough. It is estimated that gene fusions contribute to 20% of cancer incidence worldwide. Studies have shown that these events play a crucial role in the initial steps of tumorigenesis. Typical structural fusions are characteristic of tumor cells and are ideal markers for cancer cell recognition and / or targeting, such as NTRK fusions. This provides particular benefit for tumors lacking surface markers, such as triple-negative breast cancer, which accounts for 15-20% of all breast cancer cases and still lacks effective targeted therapies. Furthermore, fusion genes can provide good prognostic and / or predictive value. For example, ETV6-RUNX1 is the most common genetic abnormality, accounting for approximately 25% of all precursor B-cell lymphoblastic leukemia cases in children with acute lymphoblastic leukemia. ETV6-RUNX1 is considered an independent predictor of good prognosis, with patients having this fusion having a relatively low relapse rate.

[0004] The first evidence of gene fusions in human cancer was identified in 1960, and to date, more than 10,000 gene fusions have been discovered. This list continues to grow as research into more cancers is conducted and as technologies and bioinformatics detection methods improve. Chromosomal banding techniques in traditional cytogenetics and fluorescence in situ hybridization (FISH), gene fusion microarrays, and PCR / RT-PCR methods in molecular cytogenetics were among the first clinical diagnostic analyses used to detect fusion events. However, these methods typically have low resolution, and complex rearrangements are often difficult to detect. While cytogenetics and FISH methods remain indispensable tools for the diagnosis of fusion genes in hematological diseases and solid tumors, large-scale genome sequencing studies have recently revealed a large number of other gene fusions in cancer through a range of techniques, such as whole-genome sequencing, targeted hybridization capture, and RNA sequencing (RNA-seq), along with the development of many different bioinformatics pipelines. Among these, RNA-Seq, as an unbiased tool, facilitates the discovery and detection of fusion genes and has broad application prospects for fusion detection in clinical samples.

[0005] The general library preparation process for RNA-seq (whole transcriptome sequencing) involves extracting RNA from tumor tissue, performing rRNA removal, fragmentation, first-strand synthesis, and second-strand synthesis using dUTPs to form double-stranded DNA. A strand-specific library is then prepared through fragmentation, end repair, A base addition, adapter addition, removal of U-containing strands, and PCR amplification. After library quality control, sequencing is performed. PCR amplification not only introduces indexes (sample tags), enabling pooling and sequencing of multiple samples in the same sequencing reaction, but also affects the duplication rate (dup rate), the proportion of repetitive sequences in the total sequenced data. Too few PCR cycles result in low library concentration, failing to meet normal sequencing requirements; too many PCR cycles result in a high duplication rate, affecting quality control (QC) data. Therefore, an appropriate number of PCR cycles is crucial. Currently, the most common method used by most laboratories to determine the appropriate number of PCR cycles is based on the CT value of RT-PCR. Because the CT value represents the number of cycles required for the fluorescence signal in each reaction tube to reach a set threshold, this value indicates that the template in the tube has been sufficiently amplified but not excessively, and it has some reference value for the amplification cycle number of library PCR. However, this method adds an RT-PCR step, which not only prolongs the overall experimental time but also requires the use of corresponding materials, resulting in higher experimental costs. Furthermore, according to statistical data (see Table 1), this method cannot avoid over-amplification and inadequate amplification, and over-amplification also increases the risk of a high DUP rate.

[0006] Table 1. Methods for determining cycle number by RT-PCR and library concentration statistics.

[0007] Library concentration range (ng / μL) Percentage of Quantities >5 4.17％(5 / 120) 5-20 30.83％(37 / 120) 20-40 23.33％(28 / 120) 40-60 11.67％(14 / 120) >60 30％(36 / 120) Summary of the Invention

[0008] According to a first aspect, in one embodiment, a method for determining the number of PCR amplification cycles is provided, comprising determining the number of PCR amplification cycles for a sample according to the following formula:

[0009] The amplification cycle number y = DV200*(-4.5223) + lg(initial RNA input)*(-3.5) + K;

[0010] K is 26.7821 or 28.7821;

[0011] The amplification cycle number y is rounded to the nearest integer.

[0012] According to the second aspect, in one embodiment, a library construction method is provided, comprising:

[0013] The amplification step includes performing PCR amplification on the pretreated sample according to the number of cycles determined by any one of the methods in the first aspect, to obtain the amplified sample, which is the library.

[0014] According to the method for determining the number of PCR amplification cycles based on the above embodiments, the present invention effectively reduces the probability of over-amplification, so that a large number of samples can be within a reasonable library concentration range. Attached Figure Description

[0015] Figure 1 A graph showing the relationship between DV200, RIN values, and Mapped reads (reads aligned to the reference genome);

[0016] Figure 2 The result is for 229026662FR-labchip;

[0017] Figure 3 The result is for CR-labchip 220020961.

[0018] Figure 4 The result is for 220021353FR-labchip;

[0019] Figure 5 The result is for CR-labchip 220011551;

[0020] Figure 6 The result is 229026661FR-labchip;

[0021] Figure 7 The result is for 229026660FR-labchip. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings. In the following embodiments, many details are described to facilitate a better understanding of the present application. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other materials or methods. In some cases, certain operations related to the present application are not shown or described in the specification. This is to avoid obscuring the core parts of the present application with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; they can fully understand the related operations based on the description in the specification and general technical knowledge in the art.

[0023] Furthermore, the features, operations, or characteristics described in the specification can be combined in any suitable manner to form various embodiments. At the same time, the steps or actions in the method description can be rearranged or adjusted in a manner obvious to those skilled in the art. Therefore, the various orders in the specification and drawings are only for the clear description of a particular embodiment and do not imply a necessary order, unless otherwise stated that a particular order must be followed.

[0024] The serial numbers assigned to components in this article, such as "first" and "second", are used only to distinguish the objects being described and have no sequential or technical meaning.

[0025] In this article, "duplicate reads" refers to sequences that are obtained through sequencing and that align to the same start and end positions on the reference genome.

[0026] In this article, "dup rate" refers to the proportion of repetitive sequences in the total sequencing sequence.

[0027] In this article, "DV200" represents the percentage of RNA fragments larger than 200 nt out of all RNA fragments.

[0028] In this article, "RT-PCR" (Reverse Transcription-Polymerase Chain Reaction) refers to a technique that combines reverse transcription (RT) of RNA with polymerase chain amplification (PCR) of cDNA. In existing techniques, the number of PCR cycles is typically determined based on the CT value of RT-PCR.

[0029] To address the problems existing in the prior art, in one embodiment, the present invention provides a method for determining the number of PCR cycles for library amplification during RNA-seq library preparation of tumor samples. This method eliminates the need for RT-PCR to determine the number of library amplification cycles; the number of PCR cycles can be calculated based solely on the sample input quantity and sample quality.

[0030] To achieve the above scheme, this invention uses extensive data accumulation and simulation, assesses sample quality based on DV200 and considers sample input volume, and finally corrects the theoretical amplification cycle number based on the sample quality correction value to determine the final cycle number formula. The corrected formula effectively avoids over-amplification. For cases of insufficient amplification, this invention requires QUBIT quantification of the PCR reaction system of the library before the next purification step. Samples with a concentration less than 12 are re-batched, and subsequent purification is performed after the re-batch is completed.

[0031] According to a first aspect, in one embodiment, a method for determining the number of PCR amplification cycles is provided, comprising determining the number of PCR amplification cycles for a sample according to the following formula:

[0032] The amplification cycle number y = DV200*(-4.5223) + lg(initial RNA input)*(-3.5) + K, where K is 26.7821 or 28.7821; the amplification cycle number y is rounded to the nearest integer. The amplified product obtained from the cycle number determined by this formula, after purification, has a concentration that meets the requirements for instrumentation, effectively avoiding situations where insufficient library quantity leads to inability to be instrumented due to insufficient cycle number, or situations where a high cycle number results in only partial library selection for instrumentation, leading to missed gene detection. In one embodiment, the suitable library concentration for instrumentation (i.e., the purified concentration) is generally 5–60 ng / μL.

[0033] In one embodiment, when the DV200 of RNA in the sample is greater than 20%, the amplification cycle number y = DV200*(-4.5223) + lg(initial RNA input)*(-3.5) + 26.7821, wherein the amplification cycle number y is rounded to the nearest integer.

[0034] When the DV200 of RNA in the sample is ≤20%, the amplification cycle number y = DV200*(-4.5223) + lg(initial RNA input)*(-3.5) + 28.7821, where the amplification cycle number y is rounded to the nearest integer.

[0035] In one embodiment, after PCR amplification, if the library concentration X in the sample is ≤12 ng / μL, then amplification is performed again.

[0036] In one embodiment, if the library concentration X in the sample is greater than 12 ng / μL after PCR amplification, no further amplification is performed.

[0037] In one embodiment, after PCR amplification, if the library concentration X in the sample is 0 ≤ X < 3 ng / μL, the number of cycles for amplification is 3.

[0038] In one embodiment, after PCR amplification, if the library concentration X in the sample is 3 ≤ X < 7.5 ng / μL, the number of cycles for amplification is 2.

[0039] In one embodiment, after PCR amplification, if the library concentration X in the sample is 7.2 ≤ X < 12 ng / μL, the number of cycles for amplification is 1.

[0040] In one embodiment, the PCR amplification refers to amplification prior to hybridization capture using a probe.

[0041] In one embodiment, the PCR amplification refers to amplification prior to sequencing. For whole transcriptome sequencing, hybridization capture is not performed; sequencing can be performed after PCR amplification.

[0042] In one embodiment, after the PCR amplification, a sample is obtained that can be used for preservation or sequencing.

[0043] In one embodiment, the sample is a sample used for whole transcriptome sequencing.

[0044] In one embodiment, the sample used for amplification is a DNA sample obtained by reverse transcription of RNA.

[0045] In one embodiment, the sample is a DNA sample obtained by sequentially synthesizing RNA into one strand and then into two strands.

[0046] In one embodiment, the sample used for amplification is extracted from an autologous fluid sample or a tissue sample.

[0047] In one embodiment, the sample used for amplification is extracted from a formalin-fixed paraffin-embedded sample.

[0048] In one embodiment, the sample used for amplification is extracted from cells.

[0049] In one embodiment, the sample used for amplification undergoes the following steps sequentially: rRNA (ribosomal RNA) removal, primary purification, RNA fragmentation and denaturation, one-strand synthesis, two-strand synthesis, secondary purification, end repair and "A" addition reaction, adapter ligation, enzymatic digestion, and tertiary purification. The sample after three purifications is then used for PCR amplification.

[0050] In one embodiment, the enzymes used in the enzyme digestion step include, but are not limited to, at least one of USER enzyme, formamidopyrimidine-DNA glycosylase (Fpg), uracil-DNA glycosylase (i.e., Uracil-DNA Glycocasylase, also known as UDG enzyme), etc. This is only an exemplary list here, and other enzymes with similar functions can also be used for the enzyme digestion here.

[0051] In one embodiment, the rRNA removal includes successively undergoing probe hybridization, ribonuclease digestion (digesting the rRNA hybridized with the probe), and deoxyribonuclease digestion.

[0052] In one embodiment, the ribonuclease includes, but is not limited to, RNase H. RNase H is an endoribonuclease that can specifically hydrolyze the RNA in a DNA-RNA hybrid chain. RNase H cannot hydrolyze the phosphodiester bonds in single-stranded or double-stranded DNA or RNA, that is, it cannot digest single-stranded or double-stranded DNA or RNA.

[0053] In one embodiment, the deoxyribonuclease includes, but is not limited to, DNase I (Deoxyribonuclease I). The Chinese name of DNase I is deoxyribonuclease I, which is an endonuclease that can digest single-stranded or double-stranded DNA to produce monodeoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides.

[0054] In one embodiment, the USER enzyme creates a single nucleotide gap at the uracil position. The USER enzyme is a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII. UDG catalyzes the cleavage of the uracil base to form an abasic (apyrimidinic) site, but keeps the phosphodiester backbone structure intact. The lyase activity of Endo VIII breaks the phosphodiester bonds at the 3' and 5' ends of the abasic site, releasing the abasic deoxyribose.

[0055] In one embodiment, the probe hybridization refers to rRNA probe hybridization. The rRNA probe can be purchased from the market.

[0056] In one embodiment, it further includes purifying the amplified sample to obtain a purified sample. The purified sample can be stored after quality control. The library concentration ≥ 5 ng / μL and there is no adapter dimer contamination in the fragment quality control are considered qualified.

[0057] In one embodiment, each purification can be magnetic bead purification.

[0058] In one embodiment, the RNA starting input amount refers to the sample input amount before rRNA removal, that is, the sample input amount before rRNA probe hybridization.

[0059] According to a second aspect, in one embodiment, a library construction method is provided, including:

[0060] An amplification step, including performing PCR amplification on the preprocessed sample according to the number of cycles determined by the method of any one of the first aspect to obtain the amplified sample, which is the library. The library construction method of the present invention does not require RT-PCR and does not need to perform RT-PCR once again when determining the number of cycles as in the prior art, and a total of two RT-PCRs are required.

[0061] In one embodiment, the preprocessing includes probe hybridization, ribonuclease digestion, deoxyribonuclease digestion, primary purification, RNA fragmentation and denaturation, first-strand synthesis, second-strand synthesis, secondary purification, end repair and A-addition reaction, adapter ligation, enzyme digestion, and tertiary purification.

[0062] In one embodiment, it further includes purifying the amplified sample to obtain the purified library. The purified sample can be stored after quality control. The library concentration ≥ 5 ng / μL and the fragment quality control has no adapter dimer contamination is considered qualified.

[0063] In one embodiment, the purification can be magnetic bead purification.

[0064] According to a third aspect, in one embodiment, a library constructed by the method of any one of the second aspect is provided. The obtained library can be used for accurately detecting individual mutations in tumor samples based on next-generation sequencing technology.

[0065] Example 1

[0066] RNA samples are not as stable as DNA samples and are prone to degradation. Firstly, RNA is single-stranded, unlike the stable double-stranded structure of DNA. Secondly, the 2'-OH groups present in RNA participate in intramolecular attack on the phosphate molecules in the phospholipid bonds, leading to RNA breakage. Finally, there are many RNases in nature with high activity, easily causing RNA degradation. Therefore, in the RNA library construction process, in addition to considering the amount of RNA input, RNA quality also needs to be considered. Indicators for assessing RNA integrity include the RIN value (RNA Integrity Number) and DV200. The RIN value is a patented algorithm from Agilent Technologies for assessing RNA quality, mainly calculated by analyzing the ratio of 18S / 28S ribosomal subunits, represented by numbers 1-10. The closer the value is to 10, the higher the sample integrity; conversely, the lower the RIN value, the worse the integrity. In actual quality control, this invention found that in samples with severely degraded RNA, the 18S and 28S characteristic peaks are not obvious, resulting in a large interpretation error of the RIN, such as in FFPE samples. DV200 represents the percentage of RNA fragments longer than 200 nucleotides among all RNA fragments. Compared to assessing RNA quality in FFPE samples using RIN, samples assessed using DV200 are more likely to successfully construct libraries. This invention also presents a graph showing the relationship between DV200, RIN values, and Mapped reads (reads aligned to the reference genome). Figure 1 Analysis of DV200 and FFPE sample information quality control results showed that DV200 was correlated with mapped reads (r = 0.5798, p < 0.0001), while RIN values ​​were not correlated with mapped reads (r = 0.1762). DV200 is more suitable for assessing the degree of RNA degradation in FFPE samples. This invention not only considers the input amount of different samples but also the sample quality. It can determine the number of library amplification cycles for each sample based on the library input amount and the DV200 value, making it more scientific and effective. It can effectively avoid insufficient cycle number leading to low library concentration that cannot be sequenced, and effectively reduce the number of samples with missed gene detection due to excessive cycle number, providing stable reference data for subsequent sequencing.

[0067] According to statistical data and related reports, the number of PCR amplification cycles should be adjusted based on library construction conditions such as sample quality and input quantity. Based on these variables, the method in this embodiment mainly includes the following steps:

[0068] 1. Collect data on a large number of samples with library concentrations within a reasonable range, and compile the DV200, library preparation input, and actual PCR amplification cycles for these samples.

[0069] 2. The instructions for the relevant library preparation reagents provide different ranges of cycle numbers based on different amounts of RNA input. The corresponding regression equation derived from the amount of RNA input and the range of cycle numbers is: y = lg(RNA input) * (-3.5) + 19.167.

[0070] 3. Calculate the recommended number of PCR cycles based on the formula derived from the input amount, and then calculate the difference between the actual number of cycles and the recommended number of cycles. Finally, perform regression analysis based on the difference between the DV200 value and the above-mentioned number of cycles, and obtain the regression equation: y = DV200 * (-4.5223) + 7.6151.

[0071] 4. Finally, the formula is y=DV200*(-4.5223)+lg(RNA input)*(-3.5)+26.7821.

[0072] 5. Considering that samples with low DV200 may not be fully amplified, resulting in low library concentration, the theoretical amplification cycle number was corrected according to the sample quality correction value, resulting in the following formula:

[0073] Table 2

[0074] DV200 > 20% y = DV200 * (-4.5223) + lg(RNA input) * (-3.5) + 26.7821 DV200≤20% y = DV200 * (-4.5223) + lg(RNA input) * (-3.5) + 28.7821

[0075] 6. For cases of insufficient amplification, the PCR reaction system of the library needs to be quantified using QUBIT before the next purification step. If the concentration 0 ≤ X < 3 (ng / μL), a second amplification cycle of 3 is required; if the concentration 3 (ng / μL) ≤ X < 7.5 (ng / μL), a second amplification cycle of 2 is required; if the concentration 7.5 (ng / μL) ≤ X < 12 (ng / μL), a second amplification cycle of 1 is required; if the concentration X > 12 (ng / μL), no further amplification is needed.

[0076] 7. The overall database creation process is as follows:

[0077] 7.1.2 Probe hybridization

[0078] 7.1.2.1 Place the rRNA Depletion Solution and Probe Hybridization Buffer in an ice box beforehand. After the reagents have dissolved, vortex to mix and centrifuge. Prepare the RNA / PROBE master mix (Mix1) according to the table below, vortex to mix and centrifuge;

[0079] Table 3 Mix1 Formulation Table

[0080]

[0081]

[0082] Prepare the RNA / PROBE MASTER Mix by placing it on an ice box.

[0083] 7.1.2.2 Add 4 μL of Mix1 to 11 μL of RNA sample (the mass of RNA in the sample is the initial input amount) according to the single reaction method, mix by pipetting and centrifuging.

[0084] Because RNA is easily degraded, it should not be shaken vigorously. Instead, use a pipette to mix it, and all operations should be performed on an ice box.

[0085] 7.1.2.3 Perform hybridization on a PCR instrument according to the reaction conditions in the table below. Before incubating at 22°C, thaw the RNase H reaction buffer at 4°C.

[0086] Table 4. Conditions for rRNA probe hybridization reaction

[0087]

[0088] 7.1.2.4 After PCR incubation is complete, place the sample on ice immediately and centrifuge briefly.

[0089] 7.1.3 RNase H digestion

[0090] 7.1.3.1 After thawing, vortex the RNase H Reaction Buffer to mix well and centrifuge. After centrifuging RNase H, place it on an ice pack. Prepare the RNase H master mix (Mix 2) according to the table below, mix well by pipetting and centrifuging;

[0091] Table 5 Mix2 Preparation Table

[0092]

[0093] Prepare the RNase H master mix by placing it on an ice pack.

[0094] 7.1.3.2 Add Mix 2 to the reaction tubes in 7.1.2 in a volume of 5 μL per reaction, mix by pipetting and centrifugation.

[0095] 7.1.3.3 Place the reaction tubes on a PCR instrument and incubate at 50°C for 30 minutes, with the lid temperature set at 55°C. During incubation, thaw the DNase I Reaction Buffer at 4°C.

[0096] 7.1.3.4 After incubation, briefly centrifuge and place the sample on ice.

[0097] 7.1.4 DNase I digestion

[0098] 7.1.4.1 After thawing, vortex the DNase I Reaction Buffer and centrifuge. After centrifuging the DNase I (RNase-free), place it on an ice pack. Prepare the DNase I master mix (Mix 3) according to the table below, mix thoroughly by pipetting and centrifuging.

[0099] Table 6 Mix3 Preparation Table

[0100]

[0101] Prepare DNase I master mix by placing it on an ice pack.

[0102] 7.1.4.2 Add Mix3 sequentially to the reaction tubes in 7.1.3 according to a dosage of 30 μL for each reaction, mix by pipetting and centrifugation.

[0103] 7.1.4.3 Place the reaction tubes on a PCR instrument and incubate at 37°C for 30 minutes, with the hot cap at 45°C. During incubation, keep the RNA Sample Purification Beads at room temperature for at least 30 minutes to equilibrate.

[0104] After opening the RNA Sample Purification magnetic beads, mark the person who opened the bottle and the expiration date on the bottle. The shelf life is 6 months.

[0105] 7.1.4.4 After incubation, briefly centrifuge and place the sample on ice.

[0106] 7.1.5 Purification with magnetic beads after rRNA removal

[0107] 7.1.5.1 Prepare a 1.5 mL tube and add 90 μL of RNA Sample Purification Beads. Take 50 μL of sample from 7.1.4 and add it to the magnetic beads. Mix well by pipetting or gently shaking, and incubate on ice for 15 min to allow the magnetic beads to fully bind to the RNA fragments. During incubation, prepare 80% ethanol (10 mL DEPC water + 40 mL anhydrous ethanol) and prepare a set of eight-tube strips, labeled with serial numbers and sample names.

[0108] 7.1.5.2 After incubation, briefly centrifuge the centrifuge tube, place it on a magnetic rack until the liquid is clear and transparent, and discard the supernatant;

[0109] To reduce the risk of inter-sample contamination, only one centrifuge tube is opened at a time. The supernatant is discarded, the tube cap is closed, and then the next centrifuge tube is opened.

[0110] 7.1.5.3 Keep the centrifuge tubes on the magnetic rack and add 500 μL of 80% ethanol to each tube in sequence. After tightening the caps, invert and rinse three times, then discard the supernatant.

[0111] When adding ethanol, do not add it directly onto the magnetic bead; add it in the opposite direction to the magnetic bead's position.

[0112] 7.1.5.4 Repeat 7.1.5.3 once. After discarding the supernatant, briefly centrifuge the centrifuge tube in a handheld centrifuge, then place it on a magnetic rack. Once the solution is clear, use a 20 μL pipette to remove any remaining ethanol from the centrifuge tube.

[0113] 7.1.5.5 Drying the magnetic beads: Open the centrifuge tube and let it air dry at room temperature until the surface of the magnetic beads no longer reflects light, then close the tube cap.

[0114] The magnetic beads should be dried until the surface is no longer reflective; do not over-dry them, as this will result in product loss.

[0115] 7.1.5.6 Add 7 μL of NF water to the centrifuge tube, mix the magnetic beads with a pipette, and incubate at room temperature for 2 min.

[0116] 7.1.5.7 After incubation, briefly centrifuge the centrifuge tubes and place them on a magnetic rack until completely clear. Transfer 5 μL of the supernatant to a new eight-tube stack, discarding the magnetic bead tubes. Ensure each tube corresponds correctly during transfer to avoid confusion.

[0117] 7.2 RNA fragmentation and denaturation

[0118] Based on the extracted Q seq400 quality control results, the data was fragmented according to different DV200 values, as shown in the table below.

[0119] Table 7 Interruption Schedule

[0120] DV200 Interruption time ≥50％ 13min ＜50％ 7min

[0121] The specific operating steps are as follows:

[0122] 7.2.1 Thaw the First Strand Synthesis Reaction Buffer and Random Primers at 4°C beforehand. After the reagents have thawed, vortex to mix and centrifuge. Prepare the fragmentation and annealing mix (Mix 4) according to the table below. After vortexing and centrifuging, add 5 μL / reaction to 5 μL of purified RNA sample, and pipette up and down 10 times to mix. Place the well-mixed reaction system in a PCR instrument and incubate at 94°C for the time corresponding to Table 5. Then, immediately place on ice and perform first-strand synthesis to obtain DNA.

[0123] Table 8 Mix4 Preparation Table

[0124] Components Single reaction volume (μL) First Strand Synthesis Reaction Buffer 4 Random Primers 1 Total volume 5

[0125] 7.3 One-chain synthesis

[0126] 7.3.1 Thaw the Strand Specificity Reagent at 4°C beforehand, vortex to mix and centrifuge; centrifuge the First Strand Synthesis Enzyme Mixter. Prepare the one-chain synthesis reaction Premix (Mix5) according to the table below, vortex to mix and centrifuge, then add it to the reaction system in 7.2.1 at a ratio of 10 μL / reaction, and mix by pipetting up and down 10 times.

[0127] Table 9 Mix5 Recipe Table

[0128] Components Single reaction volume (μL) Strand Specificity Reagent 8 First Strand Synthesis Enzyme Mixter 2 Total volume 10

[0129] Premix is ​​prepared by placing it on an ice pack.

[0130] 7.3.2 Place the reaction tube from 7.3.1 into the PCR instrument and run the program in Table 10;

[0131] Table 10. Reaction conditions for one-chain synthesis

[0132]

[0133] The temperature of the hot cap needs to be ≥80℃

[0134] 7.3.3 After incubation, briefly centrifuge and place the sample tube on ice, then immediately proceed with the second-strand synthesis.

[0135] 7.4 Two-chain synthesis

[0136] 7.4.1 Thaw the Second Strand Synthesis Reaction Buffer with dUTP Mix (10×) at 4°C, vortex to mix, and centrifuge; centrifuge the Second Strand Synthesis Enzyme Mix. Prepare the second-strand synthesis Premix (Mix 6) according to the table below, vortex to mix, centrifuge, and add 60 μL / reaction to the reaction system in 7.3.1, pipetting up and down 10 times to mix. Place the well-mixed reaction system in a PCR instrument and incubate at 16°C for 1 h. After incubation, briefly centrifuge, collect the droplets on the tube wall, and immediately purify.

[0137] Table 11 Mix6 Recipe Table

[0138] Components Single reaction volume (μL) Second Strand Synthesis Reaction Buffer with dUTP Mix(10X) 8 Second Strand Synthesis Enzyme Mix 4 Nuclease-free Water 48 Total volume 60

[0139] Premix is ​​prepared by placing it on an ice pack.

[0140] 7.5 Purification after double-chain synthesis

[0141] Add 160 μL (1.8×) of purification magnetic beads to the two-chain synthesis system. For single-tube magnetic bead purification, refer to 7.5.1; for plate magnetic rack purification, refer to 7.5.2. Perform AMTK automated purification, following the purification steps outlined in JYJBJ-SOP-EI-020 "Standard Operating Instructions for Automated Purification of AMTK".

[0142] 7.5.1 Single-tube magnetic bead purification

[0143] 7.5.1.1 Add 160 μL of magnetic beads to each reaction tube, mix by pipetting or gentle shaking, and incubate at room temperature for 10 min to allow the magnetic beads to fully bind with the DNA. During incubation, prepare 80% ethanol (10 mL NF water + 40 mL anhydrous ethanol) and prepare a set of EP tubes, label them with serial numbers, print labels, and attach them to the corresponding EP tubes.

[0144] 7.5.1.2 After incubation, briefly centrifuge and place the centrifuge tube on a magnetic rack until clear;

[0145] 7.5.1.3 Discard the supernatant. To reduce the risk of intersample contamination, open only one centrifuge tube at a time, remove the supernatant, close the cap, and then open the next centrifuge tube.

[0146] 7.5.1.4 Keep the centrifuge tubes on the magnetic rack and add 500 μL of 80% ethanol to each tube. Tighten the caps, invert and rinse three times, and discard the supernatant.

[0147] Note: When adding ethanol, do not add it directly towards the magnetic bead; add it in the opposite direction to the magnetic bead's position.

[0148] 7.5.1.5 Repeat 7.5.1.4 once. After discarding the supernatant, briefly centrifuge the centrifuge tube in a handheld centrifuge, then place it on a magnetic rack. Once the solution is clear, use a 20 μL pipette to remove any remaining ethanol from the centrifuge tube.

[0149] 7.5.1.6 Drying the magnetic beads: Open the cap of the centrifuge tube and place it on a 37°C dry heat heater to dry it until the surface of the magnetic beads no longer reflects light. Then, tighten the cap and remove the centrifuge tube from the dry heat heater.

[0150] The magnetic beads should be dried until the surface is no longer reflective; do not over-dry them, as this will result in product loss.

[0151] 7.5.1.7 Add 53 μL of 0.1×TE to a centrifuge tube, mix the magnetic beads and 0.1×TE by pipetting, and incubate at room temperature for 5 min;

[0152] 7.5.1.8 After incubation, briefly centrifuge the centrifuge tubes and place them on a magnetic rack until completely clear. Transfer the supernatant to new 1.5 mL centrifuge tubes, discarding the magnetic bead tubes. Ensure that each tube corresponds correctly during the transfer to the next; do not mix them up.

[0153] 7.5.2 Plate-type magnetic bead purification

[0154] 7.5.2.1 Take a shallow well plate and add 160 μL of magnetic beads to each reaction well. Leave at least one blank row between each column of magnetic beads for gun passage.

[0155] 7.5.2.2 Add the adapter ligation reaction product to the corresponding reaction well, mix by pipetting, and incubate at room temperature for 10 min to allow the magnetic beads to fully bind to the DNA fragments. During incubation, prepare 80% ethanol (10 mL NF water + 40 mL anhydrous ethanol).

[0156] 7.5.2.3 Place the shallow-hole plate on the plate magnetic rack and let it stand for 10 minutes. After the magnetic beads have been fully attracted, discard the supernatant;

[0157] 7.5.2.4 Add 200 μL of 80% ethanol to each well, gently pipette three times, let stand for 30 seconds, and then discard the supernatant.

[0158] When adding ethanol, do not add it directly onto the magnetic bead; add it in the opposite direction to the magnetic bead's position.

[0159] 7.5.2.5 Repeat 7.5.2.4 once;

[0160] 7.5.2.6 Use a 300 μL eight-well pipette and a 10 μL eight-well pipette to aspirate the alcohol from the bottom of the shallow well plate in sequence;

[0161] 7.5.2.7 Drying the magnetic beads: Place the shallow-hole plate on a 37°C dryer and heat it until the surface of the magnetic beads no longer reflects light. Then remove it from the dryer and place it back on the plate magnetic rack.

[0162] 7.5.2.8 Add 53 μL of 0.1×TE to each well, use a pipette to mix the magnetic beads with 0.1×TE, and incubate at room temperature for 5 min to allow the DNA fragments to dissolve fully in 0.1×TE;

[0163] 7.5.2.9 Place the shallow well plate on a plate magnetic rack and let it stand for 5 minutes to fully adsorb the magnetic beads until the liquid is clear. Transfer the purified supernatant to a new eight-well plate or PCR plate and discard the shallow well plate. When transferring, pay attention to the correspondence between the two wells and do not mix them up.

[0164] 7.6 End-stage repair and adding "A"

[0165] 7.7.1 Thaw the Ultra II End Prep Reaction Buffer at 4°C. After thawing, vortex to mix and centrifuge. Centrifuge the Ultra II End Prep Enzyme Mix. Prepare the end-repair and "A" addition reaction premix (Mix 7) according to the table below. After vortexing and centrifuging, add 10 μL / reaction to the 7.5 reaction system and pipette up and down 10 times to mix. Place the well-mixed reaction system in the PCR instrument and run the program in Table 13.

[0166] Table 12 Mix7 Recipe Table

[0167] Components Single reaction volume (μL) Ultra II End Prep Reaction Buffer 7 Ultra II End Prep Enzyme Mix 3 Total volume 10

[0168] Premix is ​​prepared by placing it on an ice pack.

[0169] Table 13 End-stage repair and A-addition reaction conditions

[0170]

[0171] 7.7.2 After the reaction is complete, place the PCR tube in a handheld centrifuge, centrifuge briefly, and immediately perform adapter ligation.

[0172] 7.7 Connector Connection

[0173] 7.7.1 Thaw the Ligation Enhancer, adapter, and index at 4°C beforehand. After the reagents have dissolved, shake to mix and centrifuge for later use. Centrifuge the Ultra II Ligation Master Mix.

[0174] 7.7.2 Add 1 μL of 15 μM connector working solution to the system in 7.7.1 and mix by pipetting up and down 10 times.

[0175] 7.7.3 Prepare the reaction premix (Mix 8) according to the table below. After vortexing and centrifuging, add it to the reaction system in 7.7.2 at a ratio of 31 μL / reaction. Mix by pipetting up and down 10 times. Place the well-mixed reaction system on a constant temperature mixer at 20°C and incubate for 15 min. After incubation, briefly centrifuge to collect the liquid adhering to the tube wall.

[0176] Table 14 Mix8 Recipe Table

[0177] Components Single reaction volume (μL) Ultra II Ligation Master Mix 30 Ligation Enhancer 1 Total volume 31

[0178] 7.8 USER enzyme digestion

[0179] Add 3 μL of USER enzyme to the reaction tube in 7.7.3, vortex to mix and centrifuge, then place the reaction tube on a constant temperature mixer and incubate at 37°C for 15 min.

[0180] 7.9 Purification after connector connection

[0181] After the ligation reaction is complete, depending on the sample volume, single-tube purification, plate magnetic rack purification, and automated purification can be selected. The single-tube purification procedure is described in 7.9.1, the plate magnetic rack purification procedure is described in 7.9.2, and the AMTK automated purification procedure is described in JYJ BJ-SOP-EI-020 "AMTK Automated Purification Standard Operating Procedure".

[0182] 7.9.1 Single-tube magnetic bead purification

[0183] 7.9.1.1 Add 68 μL (0.7X) magnetic beads to each reaction tube, vortex to mix, and incubate at room temperature for 10 min to allow the magnetic beads to fully bind to the DNA fragments. During incubation, prepare 80% ethanol (10 mL NF water + 40 mL anhydrous ethanol), and prepare a set of EP tubes, label them with serial numbers, print labels, and attach them to the corresponding EP tubes.

[0184] 7.9.1.2 After incubation, briefly centrifuge the centrifuge tube, place it on a magnetic rack until the liquid is clear and transparent, and discard the supernatant;

[0185] 7.9.1.3 Keep the centrifuge tubes on the magnetic rack and add 500 μL of 80% ethanol to each tube in sequence. After tightening the caps, invert and rinse three times, then discard the supernatant.

[0186] When adding ethanol, do not add it directly onto the magnetic bead; add it in the opposite direction to the magnetic bead's position.

[0187] 7.9.1.4 Repeat 7.9.1.3 once. After discarding the supernatant, briefly centrifuge the centrifuge tube in a handheld centrifuge, then place it on a magnetic rack. Once the solution is clear, use a 20 μL pipette to remove any residual ethanol from the centrifuge tube.

[0188] 7.9.1.5 Open the cap of the centrifuge tube and place it on a 37°C dryer to dry until the surface of the magnetic bead no longer reflects light. Then, tighten the cap and remove the centrifuge tube from the dryer.

[0189] The magnetic beads should be dried until the surface is no longer reflective; do not over-dry them, as this will lead to product loss.

[0190] 7.9.1.6 Add 50 μL of 0.1×TE to the centrifuge tube, mix the magnetic beads and 0.1×TE by pipetting, and incubate at room temperature for 5 min to allow the 0.1×TE to fully dissolve the DNA fragments enriched by the magnetic beads.

[0191] 7.9.1.7 After incubation, briefly centrifuge the centrifuge tubes and place them on a magnetic rack until the solution becomes clear and transparent. Transfer the supernatant to a new centrifuge tube, discarding the magnetic bead tubes. Ensure each tube corresponds correctly during the transfer to the next; do not mix them up.

[0192] 7.9.1.8 Add 50 μL (0.9X) magnetic beads to each reaction tube, vortex to mix, and incubate at room temperature for 10 min to allow the magnetic beads to fully bind to the DNA fragments. During incubation, prepare 80% ethanol and a set of EP tubes, label them with serial numbers, print labels, and attach them to the corresponding EP tubes.

[0193] 7.9.1.9 After incubation, briefly centrifuge the centrifuge tubes and place them on a magnetic rack until the liquid is clear and transparent. Discard the supernatant.

[0194] 7.9.1.10 Keep the centrifuge tubes on the magnetic rack and add 500 μL of 80% ethanol to each tube in sequence. After tightening the caps, invert and rinse three times, then discard the supernatant.

[0195] When adding ethanol, do not add it directly onto the magnetic bead; add it in the opposite direction to the magnetic bead's position.

[0196] 7.9.1.11 Repeat 7.9.1.10 once. After discarding the supernatant, briefly centrifuge the centrifuge tube in a handheld centrifuge, then place it on a magnetic rack. Once the solution is clear, use a 20 μL pipette to remove any residual ethanol from the centrifuge tube.

[0197] 7.9.1.12 Open the cap of the centrifuge tube and place it on a 37°C dryer to dry until the surface of the magnetic bead no longer reflects light. Then, tighten the cap and remove the centrifuge tube from the dryer.

[0198] The magnetic beads should be dried until the surface is no longer reflective; do not over-dry them, as this will lead to product loss.

[0199] 7.9.1.13 Add 22 μL of 0.1×TE to the centrifuge tube, mix the magnetic beads and 0.1×TE by pipetting, and incubate at room temperature for 5 min to allow the 0.1×TE to fully dissolve the DNA fragments enriched by the magnetic beads.

[0200] 7.9.1.14 After incubation, briefly centrifuge the centrifuge tubes and place them on a magnetic rack until the solution becomes clear and transparent. Transfer the supernatant to a new centrifuge tube, discarding the magnetic bead tubes. Ensure that each tube corresponds to the previous one during the transfer to avoid confusion.

[0201] 7.9.2 Plate-type magnetic bead purification

[0202] 7.9.2.1 Take a shallow well plate and add 68 μL (0.7X) of magnetic beads to each reaction well. Leave at least one blank row between each column of magnetic beads for gun passage.

[0203] 7.9.2.2 Add the adapter ligation reaction product to the corresponding reaction well, mix by pipetting, and incubate at room temperature for 10 min to allow the magnetic beads to fully bind to the DNA fragments. During incubation, prepare 80% ethanol (10 mL NF water + 40 mL anhydrous ethanol).

[0204] 7.9.2.3 Place the shallow-hole plate on the plate magnetic rack and let it stand for 10 minutes until the magnetic beads are fully attracted, then discard the supernatant.

[0205] 7.9.2.4 Keep the shallow well plate on the magnetic rack, add 200 μL of 80% ethanol to each well, slowly blow it three times, let it stand for 30 seconds and then discard the supernatant.

[0206] When adding ethanol, do not add it directly onto the magnetic bead; add it in the opposite direction to the magnetic bead's position.

[0207] 7.9.2.5 Repeat 7.9.2.4 once.

[0208] 7.9.2.6 Use a 300 μL eight-well pipette and a 10 μL eight-well pipette to aspirate the alcohol from the bottom of the shallow well plate.

[0209] 7.9.2.7 Place the shallow-hole plate on a 37°C dryer and heat it until the surface of the magnetic beads no longer reflects light. Then remove it from the dryer and place it back on the plate-type magnetic rack.

[0210] 7.9.2.8 Add 50 μL of 0.1×TE to each well, mix the magnetic beads and TE by pipetting, and incubate at room temperature for 5 min to allow the TE to fully dissolve the DNA fragments enriched by the magnetic beads.

[0211] 7.9.2.9 Place the shallow well plate on the plate magnetic rack and let it stand for 5 minutes. After the magnetic beads are fully adsorbed, transfer the supernatant to a new shallow well plate and discard the shallow well plate used for the first purification. When transferring, make sure that each plate corresponds to the previous one and do not mix them up.

[0212] 7.9.2.10 Add 50 μL (0.9X) magnetic beads to the purified product from 7.9.2.9, mix well by pipetting, and incubate at room temperature for 10 min to allow the magnetic beads to bind to the DNA fragments. During incubation, prepare 80% ethanol (10 mL NF water + 40 mL anhydrous ethanol).

[0213] 7.9.2.11 Place the shallow-hole plate on the plate magnetic rack and let it stand for 10 minutes. After the magnetic beads are fully attracted, discard the supernatant.

[0214] 7.9.2.12 Keep the shallow well plate on the magnetic rack, add 200 μL of 80% ethanol to each well, slowly blow it three times, let it stand for 30 seconds and then discard the supernatant.

[0215] When adding ethanol, do not add it directly onto the magnetic bead; add it in the opposite direction to the magnetic bead's position.

[0216] 7.9.2.13 Repeat 7.9.2.12 once.

[0217] 7.9.2.14 Use a 300 μL eight-well pipette and a 10 μL eight-well pipette to aspirate the alcohol from the bottom of the shallow well plate.

[0218] 7.9.2.15 Place the shallow-hole plate on a 37°C dryer and heat it until the surface of the magnetic beads no longer reflects light. Then remove it from the dryer and place it back on the plate-type magnetic rack.

[0219] 7.9.2.16 Add 22 μL of 0.1×TE to each well, mix the magnetic beads with 0.1×TE by pipetting, and incubate at room temperature for 5 min to allow the TE to fully dissolve the DNA fragments enriched by the magnetic beads.

[0220] 7.9.2.17 Place the shallow well plate on a plate magnetic rack and let it stand for 5 minutes. After the magnetic beads are fully adsorbed, transfer the supernatant to a new eight-well plate or PCR plate and discard the shallow well plate. When transferring, pay attention to the correspondence between the two wells and do not mix them up.

[0221] 7.10 PCR Amplification (Non-C-PCR)

[0222] 7.10.1 Thaw the Index Primer and KAPA HiFi HotStart Ready Mix at 4°C in advance, shake to mix well and centrifuge for later use.

[0223] 7.10.2 Prepare the Non-C-PCR mix (Mix10) on ice according to Table 15.1 below. Add the Index Primer (sequences are shown in Table 15.2) (GF Primer & GR Primer) and samples separately according to the correspondence in the task sheet, so that the Index number and sample correspond one-to-one.

[0224] Table 15.1 Mix10 Formulation Table

[0225] Components Single reaction volume (μL) GF Primer (10μM) 2.5 GR Primer (10μM) 2.5 KAPA HiFi HotStart ReadyMix 25 Adapter-Ligated library 20 Total volume 50

[0226] Table 15.2

[0227]

[0228]

[0229] 7.10.3 Setting up negative and positive controls: Use TE buffer instead of the sample in the negative control wells, and add 1 μL of positive reference material to the positive control wells and bring the volume up to the nearest whole number using TE buffer. Amplify both positive and negative samples using a specially numbered Index Primer.

[0230] 7.10.4 Seal the prepared PCR reaction plate with sealing film and place it in the PCR instrument. Run the program in the table below, with a hot lid temperature of 105℃ and a 50μL system.

[0231] Table 16 NC-PCR Reaction Procedure

[0232]

[0233] The amplification cycle number in this step is simulated through the accumulation of a large amount of data, the sample quality is evaluated based on DV200, and the theoretical amplification cycle number is corrected according to the sample quality correction value to determine the final cycle number.

[0234] Table 17 NC-PCR reaction program amplification cycle number (before correction)

[0235] Formula for calculating the number of amplification cycles y = DV200 * (-4.5223) + lg(RNA input) * (-3.5) + 27.7821

[0236] Table 18 Statistical Analysis of NC-PCR Reaction Program Amplification Cycle Numbers (Before Correction) - Supplemental Amplification Sample Data

[0237] Total number of samples First round of supplementary approvals Second round of supplementary approvals 57 73.68％(42 / 57) 8.77％(5 / 57)

[0238] Table 19 Statistical Analysis of NC-PCR Reaction Program Amplification Cycle Numbers (Before Correction) - Library Concentration Data

[0239] The concentration range of the purified library (ng / μL) Percentage of Quantities 5-20 71.93％(41 / 57) 20-40 21.05％(12 / 57) 40-60 3.51％(2 / 57) >60 3.51％(2 / 57)

[0240] Table 20. NC-PCR reaction program amplification cycle numbers (corrected)

[0241]

[0242]

[0243] Table 21 Statistical Analysis of NC-PCR Reaction Program Amplification Cycle Numbers (Corrected) - Supplemental Amplification Sample Data

[0244] Total number of samples First round of supplementary approvals Second round of supplementary approvals 78 62.82％(49 / 78) 7.69％(6 / 78)

[0245] Table 22 Statistical Analysis of NC-PCR Reaction Program Amplification Cycle Numbers (After Correction) - Supplemental Amplification Sample Data

[0246] The concentration range of the purified library (ng / μL) Percentage of Quantities 5-20 62.82％(49 / 78) 20-40 25.64％(20 / 78) 40-60 7.69％(6 / 78) >60 3.85％(3 / 78)

[0247] It can be seen that by comparing Tables 21 and 22 with Tables 18 and 19, it is found that the corrected formula shows a certain reduction in both the first-round supplementary batch quantity and the second-round supplementary batch quantity, which is more convenient for experimental operators. At the same time, by statistically analyzing the concentration range of the purified library, it is found that the concentration ranges of the samples before and after correction are not significantly different. The library concentration is mainly within the range of 5 - 40 (ng / μL), and the proportion of over-amplified samples is also basically the same. Therefore, the corrected formula is used to determine the number of cycles.

[0248] Taking the starting library construction amount of 47.2 ng and the DV200 of 13.50% as an example:

[0249] Table 23 Amplification cycle number of NC-PCR reaction procedure

[0250] Initial database size (ng) DV200 PCR cycles formula 47.2 13.50％ 22 y = DV200 * (-4.5223) + lg(RNA input) * (-3.5) + 28.7821

[0251] 7.11 Non-C-PCR product purification

[0252] 7.11.1 According to the sample volume, single-tube purification, plate magnetic stand purification, and automated purification can be selected.

[0253] 7.11.2 Use 45 μL (0.9×) magnetic beads to purify the PCR product, and finally dissolve it in 22 μL of TE (pH 8.0). The operation process refers to 7.5. After purification, transfer the purified product to a pre-prepared 1.5 mL centrifuge tube, and transfer them in corresponding order.

[0254] 7.12 Library quality control

[0255] 7.12.1 After measuring the total library amount of the completed library samples using Qubit or microplate reader, send them for inspection. The specific operation steps are detailed in JYJBJ-SOP-EI-030 "Standard Operating Procedure for the Use and Maintenance of Invitrogen Qubit 3.0 Fluorescence Quantification Instrument" and JYJBJ-SOP-EI-024 "Standard Operating Procedure for BioTek Multifunctional Microplate Reader". The library concentration ≥ 5 ng / μL and the fragment quality control without adapter dimer contamination are considered qualified.

[0256] Library concentration < 5 ng / μL belongs to unqualified samples, and arrange for re-library construction or send an unqualified notice.

[0257] 7.12.2 Only when both the negative control and the positive control are qualified can subsequent tests be carried out. The purified concentration of the negative control is 0 - 1 ng / μL, and the purified concentration of the positive control is 10 - 30 ng / μL.

[0258] 7.13 Sample storage

[0259] Store the completed library samples at -25 to -15 °C; store them in the sample center within one week.

[0260] 8. Database Construction Results

[0261] Table 24

[0262]

[0263] Taking sample 229026662FR as an example, the theoretical PCR amplification cycle number y = 0.514*(-4.5223) + lg(100)*(-3.5) + 28.7821 = 0.514*(-4.5223) + 2*(-3.5) + 28.7821 ≈ 17. The same logic applies to other samples; the calculated theoretical PCR amplification cycle number is rounded to the nearest integer.

[0264] Labchip results are as follows Figures 2-7 As shown, where, Figure 2 The result is 229026662FR-labchip. Figure 3 The result is for CR-labchip 220020961. Figure 4 The result is for 220021353FR-labchip. Figure 5 The result is for CR-labchip 220011551. Figure 6 The result is 229026661FR-labchip. Figure 7 The result is for 229026660FR-labchip.

[0265] The results in Table 23 show that the difference between the theoretical cycle number calculated using the formula provided in this embodiment and the actual cycle number is not significant. Furthermore, the concentrations of the library samples amplified using this formula, and even those supplemented according to the supplementary batching rules, are within reasonable ranges, and no abnormal library concentrations have been observed. Additionally, observation of the Labchip images of these samples... Figure 2-7 It can be observed that the main fragment sizes of these libraries are all within a suitable range, and there are no obvious abnormal peaks, i.e., abnormal fragment sizes.

[0266] For the number of cycles, if the number of additional batches is relatively large, the main impact is to increase the experimental time.

[0267] The purified library can be used for sequencing when the concentration is 5–60 ng / μL.

[0268] The batch replenishment rule targets the concentration before purification. The concentration determination before purification uses the entire unpurified sample after amplification. Some substances present in the PCR system (such as excessive index, adapters, etc.) can lead to an inflated concentration, and some loss also occurs during subsequent purification. Therefore, the batch replenishment rule is set up.

[0269] In one embodiment, the present invention omits the RT-PCR step in the library construction process, simplifying the operation procedure, effectively saving time and reducing experimental costs. The present invention uses a conventional PCR amplification method, omitting RT-PCR.

[0270] In one embodiment, the present invention effectively reduces the probability of over-amplification, so that a large number of samples can be within a reasonable library concentration range.

[0271] In one embodiment, the present invention employs a pre-purification batch replenishment method for insufficiently amplified samples, effectively avoiding post-purification batch replenishment, significantly shortening the experimental time, and reducing experimental costs.

[0272] In one embodiment, the present invention determines the formula for the final PCR amplification cycle number based on a large amount of data accumulation, DV200, and sample input. This simplifies the library preparation experimental procedure and reduces experimental costs.

[0273] In one embodiment, the present invention provides the following two formulas for calculating the number of PCR cycles.

[0274] Table 25

[0275]

[0276] The above examples illustrate the present invention only to aid in understanding it and are not intended to limit the scope of the invention. Those skilled in the art can make various simple deductions, modifications, or substitutions based on the principles of this invention.

Claims

1. A method of determining the number of PCR amplification cycles, characterized by, This includes determining the number of PCR amplification cycles for the sample used for amplification using the following formula: The amplification cycle number y = DV200 * (-4.5223) + lg(initial RNA input) * (-3.5) + K; The amplification cycle number y is rounded to the nearest integer. K is 26.7821 or 28.7821; when the DV200 of RNA in the sample is >20%, the amplification cycle number y = DV200*(-4.5223) + lg(initial RNA input)*(-3.5) + 26.7821; when the DV200 of RNA in the sample is ≤20%, the amplification cycle number y = DV200*(-4.5223) + lg(initial RNA input)*(-3.5) + 28.7821; in: The initial RNA input is measured in ng. The PCR amplification refers to the amplification prior to hybridization capture using probes, and the PCR amplification refers to the amplification prior to sequencing. After the PCR amplification, a sample for whole transcriptome sequencing is obtained that can be preserved or sequenced; that is, the amplified sample is obtained. DV200 represents the percentage of RNA fragments larger than 200 nt in a sample. If the library concentration X in the sample is ≤12ng / μL after PCR amplification, then amplification should be performed again. If the library concentration X in the sample is greater than 12 ng / μL, then no further amplification will be performed; If the library concentration X in the sample is 0 ≤ X < 3 ng / μL, the number of cycles for amplification is 3. If the library concentration X in the sample is 3 ≤ X < 7.5 ng / μL, the number of cycles for amplification is 2. If the library concentration X in the sample is 7.5 ≤ X < 12 ng / μL, the number of cycles for amplification is 1.

2. The method of claim 1, wherein, The sample used for amplification is a DNA sample obtained by reverse transcription of RNA; Alternatively, the sample used for amplification is a DNA sample obtained by sequentially synthesizing RNA into one strand and then into two strands. Alternatively, the sample used for amplification may be an autologous fluid sample or a tissue sample; Alternatively, it may also include purifying the amplified sample to obtain a purified sample.

3. A library construction method characterized by, include: The amplification step includes performing PCR amplification on the sample to be amplified according to the number of cycles determined by the method according to claim 1 or 2, to obtain the amplified sample, which is the library.

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