Full-length lncrna absolute quantitative transcriptome library construction and sequencing method based on tso-umi marker and composite internal standard correction

Abstract

The application provides a full-length lncRNA absolute quantitative transcriptome library construction and sequencing method based on TSO-UMI marking and composite internal standard correction, and the library construction method steps comprise the following steps: S1, total RNA of a sample is extracted and rRNA is removed; S2, a composite internal standard is added; S3, a reverse transcription adapter primer is used to connect a product; S4, a TSO-UMI fusion primer is added, and full-length cDNA is synthesized through template switching; S5, cDNA is used as a template to perform PCR amplification; and S6, the amplified library is purified; the nucleotide sequence of the TSO-UMI fusion primer is shown in SEQ ID NO. 1 or SEQ ID NO. 2. Through TSO-UMI fusion primer sequence reconstruction, SIRV / ERCC specific composite internal standard ratio, full-process coupling experimental design, and supporting UMI+internal standard joint correction integrated bioinformatics algorithm, the problems such as PCR deviation, relative quantification, batch difference, isomer recognition, and polyA-free quantification are successfully solved, and reliable technical guidance is provided for lncRNA basic research, disease marker screening, and clinical efficacy monitoring.

Description

Technical Field

[0001] This invention relates to the field of long non-coding RNA sequencing technology, specifically to a method for absolute quantitative transcriptional library assembly and sequencing of full-length lncRNA based on TSO-UMI labeling and compound internal standard correction. Background Technology

[0002] Long non-coding RNAs (lncRNAs) are >200 nt in length and have no protein-coding ability, but they are widely involved in core biological processes such as chromatin remodeling, transcriptional regulation, mRNA degradation, cell proliferation and differentiation, and malignant tumor progression. These RNAs have three main characteristics: generally low expression abundance, a large number of alternative splicing isoforms, and most lncRNAs do not have a PolyA tail. Traditional second-generation short-read sequencing and conventional third-generation long-read sequencing both suffer from drawbacks such as quantitative distortion, incomplete transcript recognition, and difficulty in isoform differentiation.

[0003] The prior application (CN202410049819.0) has established a technical system for rRNA removal + 3' end specific adapter ligation + template switching reverse transcription (TSO) + ONT long read full-length library construction, successfully solving the following key pain points: 1. Second-generation short-read length splicing errors, isomer confusion, and inaccurate quantification; 2. Conventional third-generation sequencing showed PolyA tail loss and no missed detection of PolyA lncRNA; 3. Achieve full-length capture, preserve PolyA length, and simultaneously cover both mRNA and lncRNA.

[0004] However, several key limitations still exist: 1. PCR amplification bias: During library construction, PCR can lead to differences in amplification efficiency of different transcripts, resulting in excessively high quantitative CV values ​​and distortion of low-abundance disease-related lncRNA signals; 2. It can only output relative quantitative indicators such as TPM (Transcripts Per Million) and FPKM (Fragments Per Kilobase of transcript per Million mapped reads). The quantitative results are affected by the amount of sequencing data, sample quality differences, and batch operation differences. It cannot obtain the true copy number of the original RNA molecule, and cross-sample and cross-batch data are not comparable. 3. The lack of standardized correction internal standards adapted to the high heterogeneity of lncRNAs makes it impossible to distinguish the true expression levels of different alternative splicing isoforms, and isoform quantifications are mutually confusing; 4. There is no dedicated correction system for lncRNAs that do not contain PolyA type, and the expression levels of these key transcripts may be generally underestimated.

[0005] There are currently two mainstream technologies in the industry for transcriptome quantitative correction: UMI single-molecule labeling technology and Spike-in exogenous internal standard correction technology. At the same time, TSO template switching technology is used for full-length cDNA synthesis. However, the three technologies, whether used independently or simply spliced ​​together, cannot be adapted to the ONT full-length lncRNA sequencing scenario, and there are multiple technical contradictions.

[0006] For the second-generation platform UMI+Spike-in combined quantitative scheme, UMI (unique molecular identifier) ​​is a random or specific short nucleotide sequence, usually 6-10 bases in length, which can be linked to each DNA molecule during library construction. Its core principle is to add a unique tag to each original molecule before PCR amplification. After amplification and sequencing, readings with the same UMI are considered to originate from the same original molecule, while readings with different UMIs represent different molecules. This can distinguish between PCR duplications and true variations, and improve the sensitivity of low-frequency variation detection.

[0007] Spike-in refers to a pre-designed reference that can be directly added to the sample to be tested and used in library construction, sequencing, and other processes. These reference sequences are often non-human sequences or artificially synthesized sequences. After sequencing, the sequence characteristics can be used to determine which sequences are reference sequences and which are the sample to be tested.

[0008] The second-generation platform's UMI+Spike-in combined quantification scheme is only compatible with Illumina short-read sequencing. Before sequencing, RNA fragmentation is required, making it impossible to obtain the full-length lncRNA sequence naturally. It also loses PolyA tail length information and cannot capture PolyA-free lncRNA. Its UMI tag is attached to the end of the universal sequencing adapter. If it is directly grafted into the TSO reverse transcription system, it will disrupt the TSO 3' end rG triplet template switching structure, resulting in a significant decrease in template switching efficiency and a noticeable decrease in full-length capture rate. Using only Spike-in as an internal standard, it lacks isoform components that simulate alternative splicing and ultra-long transcript internal standards. It cannot correct for quantitative biases caused by lncRNA isoforms and long RNA fragments, and can only achieve relative correction at the gene level, failing to achieve absolute quantification of isoforms.

[0009] Conventional UMI-RNAseq technology relies on the basic principle of UMI to eliminate PCR amplification duplications. UMI sequences are all designed with pure random bases and no error-correcting separator bases. It is adapted for short-read sequencing, but does not incorporate a TSO template switching mechanism and is not compatible with long-read full-length library construction. It lacks a compound gradient exogenous internal standard correction process. Relying solely on UMI can only remove PCR duplications and cannot offset systematic errors caused by reverse transcription, adapter ligation, sequencing, and sample batches, thus failing to achieve true copy number output.

[0010] In addition, there is the TSO template switching reverse transcription technology. The existing TSO is only used in sequencing scenarios that rely on Oligo (dT) to enrich PolyA+ mRNA, and is completely incompatible with PolyA-free lncRNA capture. It only carries a simple 6-10nt cell barcode and does not have error-correcting UMI single-molecule labeling function. The original bases of ONT long-read sequencing have a certain error rate, and ordinary uncorrecting barcodes will cause a large number of UMI sequences to be mis-clustered, resulting in distortion of low-abundance transcript quantification. There is no matching SIRV / ERCC composite internal standard correction system, and it is not compatible with the prior patented 3' end-specific lncRNA capture adapter system, and cannot meet the lncRNA full-length quantification needs of batch tissue and clinical micro-samples.

[0011] The basic functions of UMI, TSO, and ERCC / SIRV are known in this field, but directly splicing the three together for ONT full-length lncRNA sequencing will result in three irreconcilable technical contradictions: 1) Molecular structure conflict: Insertion of ordinary random UMI sequences into TSO primers will disrupt the 3' rG triplet hybridization structure, reduce template switching efficiency by more than 30%, and significantly reduce the full-length effective reads; 2) Sequencing platform compatibility conflict: ONT long reads have an inherent base error rate, and pure random UMI without error correction will have a large number of incorrect matches, resulting in distortion of single molecule counts; 3) Internal standard adaptation conflict: A single ERCC only covers a limited length and a single transcript structure, which cannot match the triple complexity of lncRNA multiple isoforms, ultra-long fragments, and no PolyA structure. The calibration curve fit is poor and the quantification error exceeds 20%.

[0012] In summary, developing an integrated library preparation and sequencing solution that is compatible with prior patent advantages, eliminates the mutual contradictions among the three types of technologies, eliminates PCR bias, achieves absolute quantification, is compatible with the ONT long read platform, and obtains the true copy number output of lncRNA isoforms is of great value and significance. Summary of the Invention

[0013] This invention proposes a method for absolute quantitative transcriptional library assembly and sequencing of full-length lncRNA based on TSO-UMI labeling and compound internal standard correction, in order to at least solve one of the many problems in the prior art.

[0014] The technical solution of this invention is implemented as follows: The first aspect of this invention is to provide a method for absolute quantification of full-length lncRNA transcriptomes based on TSO-UMI markers and compound internal standard correction, comprising the following steps: S1. Extract total RNA from the sample and remove rRNA; S2. Add SIRV / ERCC composite internal standard; S3. Ligate the product from step S2 using reverse transcription adapter primers; S4. Add the TSO-UMI fusion primer and synthesize full-length cDNA by template switching; S5. Perform PCR amplification using cDNA as a template; S6. The amplified library was purified to obtain a full-length lncRNA absolute quantitative transcriptome library; The nucleotide sequence of the TSO-UMI fusion primer is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0015] Furthermore, the total RNA purity satisfies an A260 / A280 ratio of 1.9-2.1 and an integrity RIN ≥ 8.0; and / or, the rRNA is removed to a purity of ≥ 95%.

[0016] Furthermore, the composite internal standard is SIRV-Set 4 from Lexogen, added at an amount of 0.5-1% of the total RNA.

[0017] Furthermore, the reverse transcription adapter primers shown include primers with nucleotide sequences as shown in SEQ ID NO. 8-14.

[0018] Furthermore, sequencing barcodes are added to the PCR amplification process.

[0019] Furthermore, the number of PCR amplification cycles is ≤13.

[0020] The second aspect of the present invention is to provide a full-length lncRNA absolute quantitative sequencing library, which is obtained by the library construction method described in the first aspect.

[0021] The third aspect of this invention is to propose a method for absolute quantitative transcriptome sequencing of full-length lncRNA, comprising loading the sequencing library described in the second aspect into an ONT sequencing chip, performing long-read sequencing to obtain a sequence covering the complete transcript, filtering the sequence, and performing lncRNA quantitative analysis based on UMI and internal standards.

[0022] Furthermore, the quantitative analysis includes: a. Obtain the original number of molecules by removing duplicates using UMI clustering; b. Screen full-length reads to identify lncRNAs and their isoforms; c. Count the number of reads of the internal standard, correct the endogenous RNA according to the internal standard, and output the true copy number; d. Differentiate and output the absolute expression levels of each lncRNA isoform.

[0023] Preferably, the calibration of the endogenous RNA is performed based on the standard curve method using internal standards.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: The library construction method described in this invention overcomes multiple technical obstacles by designing TSO-UMI fusion primer sequence reconstruction, SIRV / ERCC-specific compound internal standard ratios, a fully coupled design, and a matching UMI + internal standard joint correction integrated bioinformatics algorithm. It successfully solves industry pain points such as PCR bias, relative quantification, batch differences, isoform identification, and quantification without polyA. Sequencing results demonstrate stable full-length rate, accurate quantification, clear isoform resolution, and wide applicability. It provides high-precision, quantifiable, and comparable technical support for lncRNA basic research, disease biomarker screening, and clinical efficacy monitoring, possessing significant industrialization value. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] This invention provides a method for absolute quantitative transcriptional library construction of full-length lncRNA based on TSO-UMI labeling and compound internal standard correction. Building upon the method described in previous patent application (CN202410049819.0) involving rRNA removal, 3' end-specific adapter ligation, TSO template replacement, and ONT long-read library construction, the following improvements are made: 1) TSO 5' end fusion error-correcting UMI: The TSO primer backbone is fixed and the 3' upstream spacer is modified into an error-correcting UMI. While retaining the core rG structure of template switching, an error-correcting molecular tag is embedded, which balances full-length capture efficiency and single-molecule labeling function. An error-correcting UMI with spacer-distinguishing bases is designed to reduce UMI error clustering caused by ONT sequencing error, stably restore the number of original RN molecules without affecting the TSO secondary structure, do not reduce the full-length rate, achieve single-molecule unique labeling, and eliminate PCR amplification bias. 2) Precise incorporation of the SIRV / ERCC composite internal standard: Lexogen's SIRV-Set 4 was used as the composite internal standard, containing the following three components: SIRV Isoforms: This module contains 69 artificial transcription variants that mimic the splicing characteristics of 7 human model gene loci; it also includes additional isoforms and transcription variants to fully reproduce various forms of transcriptional variation in organisms: alternative splicing, alternative transcription start sites, alternative transcription stop sites, overlapping gene transcripts, and antisense transcripts.

[0027] ERCC: This module contains a total of 92 artificial transcripts as mixed standards, strictly following the design logic of 1 gene = 1 exon = 1 transcript. Each ERCC transcript has a unique sequence identifier. This set of mixed standards with all single isoforms can intuitively complete three types of experimental evaluations: dose response curve fitting, limit of detection (LOD) determination, and efficiency evaluation of the complete library construction process.

[0028] Long SIRV: This module features five length gradients (4 kb, 6 kb, 8 kb, 10 kb, and 12 kb), each containing three independent transcripts, for a total of 15 artificial RNAs. This series of length gradients covers the vast majority of endogenous cellular transcripts; all 15 long SIRV sequences are unique and do not overlap with other exogenous internal standards or endogenous cellular transcripts; the equimolar ratio of long SIRV sequences makes it the optimal tool for assessing transcript length preferences in RNA sequencing workflows.

[0029] The combined module SIRV-Set 4 serves as an internal standard, covering the triple complexity of isoform diversity, abundance dynamic range, and length span. It has no homology with the human / mouse genome and does not interfere with the detection of endogenous RNA.

[0030] 3) Integrated algorithm for UMI deduplication + internal standard curve + full-length isomer absolute quantification: realizes single-molecule correction, batch correction, isomer resolution, and true copy number output.

[0031] Specifically, to meet the special requirements of full-length lncRNA capture, UMI labeling, and ONT long-read platforms, this invention optimizes the structure of template substitution oligonucleotides (TSO) and designs multiple sets of differentiated TSO-UMI fusion primer sequences.

[0032] The TSO-UMI fusion primer includes a fixed 5' end conserved backbone and a downstream UMI region. The fixed 5' end conserved backbone sequence is: 5'-CTGTTGGTGCTGATATTGCT-3. The downstream UMI region is optimized through rearrangement, spacing, and length combinations to improve specificity, UMI compatibility, and polyA-RNA-free capture efficiency.

[0033] As an alternative, seven differentiated TSO-UMI fusion primer sequences were designed as shown in Table 1 below.

[0034] Table 1:

[0035] Of the seven sequences above, sequence 1 performs exceptionally well in conventional full-length mRNA sequencing, while sequence 2 is specifically adapted for lncRNA and high-precision quantification scenarios. Each sequence has its own strengths and complements the others.

[0036] Specifically, the method for absolute quantitative transcriptional assembly and sequencing of full-length lncRNA includes the following steps: 1) Sample RNA extraction and quality control Total RNA was extracted from samples such as cells and tissues, and its concentration, purity (A260 / A280=1.9-2.1) and integrity (RIN≥8.0) were tested to ensure that the RNA quality met the requirements for full-length library construction.

[0037] 2) Highly efficient removal of rRNA

[0038] Using an rRNA removal kit, more than 95% of ribosomal RNA was removed through probe hybridization, RNase H digestion, and DNase I treatment, while retaining effective transcripts such as mRNA, lncRNA, and circRNA. The transcripts were then purified and recovered using magnetic beads.

[0039] 3) Precise incorporation of SIRV / ERCC / composite internal standard

[0040] Take 10-50 ng of rRNA deplete-RNA, add serially diluted SIRV / ERCC internal standard, and finally the proportion of total RNA should be 0.5-1%. Mix thoroughly to allow the internal standard and endogenous RNA to be co-processed, co-constructed into a library, and co-sequencing, which will serve as the basis for subsequent quantification.

[0041] 4) 3' end special connector connection

[0042] Using reverse transcription adapter primers such as Lnc-Pm-Cm, Lnc-Pm-1, Lnc-Pm-2, Lnc-Pm-3, Lnc-Pm-5, Lnc-Pm-10, and Lnc-Pm-20, rRNA-depleted RNA is annealed to the 3' reverse transcription adapters of the Lnc-Pm series designed in the prior patent. The 3' end specific ligation is completed by T4 ligase catalyzing the process, providing a fixed start end for subsequent reverse transcription.

[0043] The reverse transcription adapter primer sequences are shown in Table 2.

[0044] Table 2:

[0045] Lnc-Pm-Cm was synthesized using modified bases, with phosphorylation modification of the 5' G end and 3' NH2C6 modification of the 3' A end. Lnc-Pm-1 was synthesized using modified bases, with phosphorylation modification applied to the 5' end T. Lnc-Pm-2 was synthesized using modified bases, with phosphorylation modification applied to the 5' end T. Lnc-Pm-3 was synthesized using modified bases, with phosphorylation modification at the 5' end T. Lnc-Pm-5 was synthesized using modified bases, with phosphorylation modification applied to the 5' end T. Lnc-Pm-10 was synthesized using modified bases, with the 5' end T modified by phosphorylation.

[0046] 5) TSO-UMI template switching reverse transcription

[0047] A TSO-UMI fusion primer was added, with a random UMI+ error-correcting base at the 3' end and a fixed sequence at the 5' end; full-length cDNA was synthesized through a template switching mechanism, with each original RNA molecule being uniquely labeled with a UMI, and the full-length ratio was maintained at ≥80%.

[0048] 6) High-fidelity amplification of full-length cDNA

[0049] Long-fragment high-fidelity DNA polymerase was used to amplify full-length cDNA, and sequencing barcodes were introduced to obtain a full-length library with UMI and barcodes. The amplification cycle was strictly controlled to not exceed 13 cycles to avoid over-amplification and deviation.

[0050] 7) Library purification and quality control

[0051] Magnetic beads are used to purify and remove short fragments, primer dimers, and non-specific products; library concentration and fragment distribution are detected, and qualified libraries have a length ≥1 kb and a concentration ≥10 ng / μL.

[0052] 8) ONT long read sequencing

[0053] The qualified library was loaded into the ONT sequencing chip for long-read sequencing to obtain full-length reads with an N50 length ≥ 1 kb, covering the complete transcript sequence.

[0054] 9) UMI + Internal Standard Combined Absolute Quantitative Analysis

[0055] ① Raw data quality control: Filtering low-quality reads and removing connectors; ②UMI clustering for deduplication: Using UMI + alignment position as the unique identifier, PCR duplicates are removed to restore the original number of molecules; ③ Full-length transcript identification: Screen full-length reads to identify lncRNAs and their isoforms; ④ Internal standard quantification: Count the number of SIRV / ERCC reads and fit a standard curve (R²). 2 ≥0.99); ⑤ Absolute Quantitative Correction: Correct endogenous RNA reads based on standard curves, outputting the true copy number in the copy / ng level; ⑥ Isoform quantification: Differentiate different lncRNA isoforms and output the absolute expression level of each isoform.

[0056] Compared to existing technologies, the above library preparation and sequencing methods have the following advantages.

[0057] 1) It retains all the advantages of full-length capture, PolyA tail length preservation, and simultaneous detection of mRNA and non-PolyA lncRNA; 2) Solving the problem of mutual exclusion between UMI and TSO structures: The optimized fusion primers do not affect the template switching efficiency and the full length ratio is stable at ≥80%, avoiding the defect of the ordinary splicing scheme with a full length ratio of less than 70%; 3) Eliminating PCR amplification bias: UMI single-molecule labeling and deduplication correction reduced the quantitative CV value from 10–20% to ≤10%, and low-abundance lncRNA signals were stable and reproducible; 4) Achieve absolute quantification of full-length lncRNA isoforms for the first time: Using SIRV / ERCC composite internal standard, output the true copy number at the copy / ng level with an error of ≤12%, solving the industry pain point of long-term reliance on relative quantification; 5) Full-dimensional internal standard correction: Simultaneously covers the triple complexity of isoform diversity, abundance dynamic range, and length span, adapting to the highly heterogeneous characteristics of lncRNA; 6) No reduction in overall length ratio: The UMI fusion design does not affect TSO efficiency, and the UMI splitting ratio is ≥75%; 7) More accurate quantification of PolyA-free lncRNAs: Without relying on PolyA enrichment and internal standard correction to eliminate bias, the accuracy of quantification of these key lncRNAs is greatly improved; 8) Cross-sample / batch comparability: Internal standard correction of batch, sequencing depth, and sample quality differences significantly enhances result stability and comparability; 9) Significantly expanded applicable scenarios: Suitable for high-precision research and applications such as micro-samples, clinical tissues, disease biomarker screening, prognostic assessment, and efficacy monitoring; 10) High technical barriers: The TSO primer backbone is fixed and the 3' upstream spacer is modified into an error-correcting UMI. While retaining the core rG structure of template switching, an error-correcting molecular tag is embedded. An error-correcting UMI with spacer-distinguishing bases is designed. Through TSO-UMI fusion + SIRV / ERCC composite internal standard + full-length absolute quantification, it is the first of its kind in the industry and has significant technical advantages and application value.

[0058] Example 1: Performance Verification of Different TSO Sequences

[0059] I. RNA extraction from the sample to be tested

[0060] 1. Select cryopreserved 293T cell material and rapidly grind it in liquid nitrogen; 2. Grind into powder and transfer to a 2ml EP tube at low temperature; 3. Add Trizol (1.0 mL) lysis buffer, 20 μL mercaptoethanol, and 10 μL proteinase K, mix thoroughly, and place on ice for 10 minutes; 4. After pyrolysis, at 4℃ and 12000g / 5min, transfer the supernatant to a new EP tube; 5. Transfer the supernatant to a new centrifuge tube, add 200 μL of chloroform, vortex to mix, incubate at 4°C for 12000 g / 10 min, and then transfer the supernatant to a new EP tube. 6. Add twice the volume of CTAB lysis buffer, add urea to a final concentration of 2M, add LiCl to a final concentration of 0.6M, and add Triton X-100 to a final concentration of 0.5%; 7. After placing on ice for 10 minutes, incubate at 50°C for 10 minutes; 8. Add a certain volume of chloroform at a ratio of (1000:200), vortex mix, 4℃, 12000g / 10min, and transfer the supernatant to a new EP tube; 9. Add 200 μL of isopropanol and place on ice to precipitate for 30 minutes; 10.4℃, 12000g / 10min, discard the waste liquid and keep the precipitate, add 1mL of 75% ethanol, and gently invert to completely suspend the precipitate; 11. Repeat the above steps twice; 12. Depending on the size of the precipitate, add an appropriate amount of enzyme-free water to dissolve the RNA; Transfer RNA to a new 1.5ml EP tube at 13.4℃ and 12000g for 2 minutes. 14. Use Nanodrop to measure its absorbance and A260 / 280 and A260 / 230; 15. Use Qubit to detect its concentration and calculate the total amount based on the volume; 16. Use agarose gel electrophoresis to detect the degree of RNA degradation or Agileng 2100 to detect its RIN value; 17. After testing, freeze at -80℃ for subsequent use. Take RNA with purity (A260 / A280=1.9-2.1) and integrity RIN value ≥8.0 for full-length library construction.

[0061] II. Sample rRNA Removal (Ribo-off rRNA Depletion Kit (Human / Mouse / Rat))

[0062] 1. In a Nuclease-free PCR tube, dilute 0.1–1 μg of total RNA to 11 μl with Nuclease-free ddH2O and store on ice for later use; 2. Prepare the following reaction solution in a nuclease-free PCR tube:

[0063] 3. Gently pipette 10 times to mix thoroughly, then briefly centrifuge to collect the mixture at the bottom of the tube; 4. Place the sample in a PCR instrument and incubate at 68℃ for 10 min to perform probe hybridization:

[0064] 5. Briefly centrifuge to collect the sample to the bottom of the tube, place it on ice, and immediately proceed to the next step; 6. Prepare the following reaction solution on ice for RNase H digestion:

[0065] 7. Gently pipette to mix, then briefly centrifuge to collect the sample at the bottom of the tube; 8. Place the sample in a PCR instrument and react at 37°C for 30 min; 9. Briefly centrifuge to collect the sample to the bottom of the tube and place it on ice. Proceed to the next step immediately.

[0066] 10. Prepare the following reaction solution on ice for DNase I digestion:

[0067] 11. Gently pipette to mix, then briefly centrifuge to collect the sample at the bottom of the tube; 12. Place the sample in a PCR instrument and react at 37°C for 30 min; 13. After a brief centrifugation, collect the sample to the bottom of the tube and place it on ice. Proceed immediately to the next step.

[0068] III. Purification of Ribosomal-depleted RNA using VAHTS RNA Clean Beads

[0069] 1. Vortex to mix VAHTS RNA Clean Beads. Add 110 μl (2.2 ×) to the RNA sample from the previous step and pipette 10 times to mix thoroughly. 2. Let stand on ice for 15 minutes to allow the RNA to bind to the magnetic beads; 3. Let stand on the magnetic rack for 5 minutes until the solution becomes clear, then carefully remove the supernatant; 4. Keep the sample on the magnetic rack at all times, add 200 μl of freshly prepared 80% ethanol with Nuclease-free ddH2O, rinse the magnetic beads (be careful not to blow the magnetic beads apart), incubate at room temperature for 30 seconds, and carefully remove the supernatant; 5. Repeat the previous step, rinsing a total of 2 times. 6. Keep the sample on the magnetic rack at all times, and dry the magnetic beads at room temperature for 5-10 minutes with the lid off; 7. Remove the sample from the magnetic rack, add 10.5 μl of Nuclease-free ddH2O, and mix thoroughly by pipetting 6 times. Let stand at room temperature for 2 min. Let stand on the magnetic rack for 5 min until the solution is clear, then carefully aspirate 8 μl of supernatant into a new Nuclease-free PCR tube; 8. Samples should be immediately used for RNA-seq library construction or other analytical applications, or stored at -80 to -65°C.

[0070] IV. Reverse Transcription Adapter Ligation

[0071] 1. Annealing of reverse transcription adapters and Ribosomal-depleted RNA, reaction system:

[0072] 2. Gently pipette to mix, then briefly centrifuge to collect the sample at the bottom of the tube; React at 3.65°C for 5 minutes, then place on ice to cool rapidly; 4. Joint connection reaction, add the following reaction system:

[0073] 5. Gently pipette to mix, then briefly centrifuge to collect the sample at the bottom of the tube; React at 6.24°C for 30 min, mixing once by blowing during the process.

[0074] V. Reverse transcription reaction generates cDNA

[0075] 1. Reverse transcription reaction, add the following reaction system:

[0076] 2. Gently pipette to mix, then briefly centrifuge to collect the sample at the bottom of the tube; React at 3.42°C for 2 minutes; 4. Add 2 µl of Maxima H Minus Reverse Transcriptase. Total volume: 60 µl; 5. Gently pipette to mix, then briefly centrifuge to collect the sample at the bottom of the tube; React at 6.42°C for 90 minutes. VI. Amplification Reaction 1. Prepare the following amplification reaction system:

[0077] 2. Amplification procedure:

[0078] 7. Purify using 0.9X Ampure XP beads; 1. Use 0.9X Ampure XP beads to purify and remove short fragments, primer dimers, and non-specific products; 2. Elute cDNA with 24 μL NF-κB water to construct the ONT library; 8. Mixing PCR products The amplified products were mixed in equal amounts, with a mixed sample volume not exceeding 400 ng and a mixed sample volume of 20 μL.

[0079] 9. Constructing libraries and sequencing using PCR mixture products to ligate sequencing adapters.

[0080] 1. Perform subsequent repair, adapter addition, and purification processes according to the ONT cdna procedure.

[0081] 2. Load the library into the ONT sequencing chip and perform long-read sequencing to obtain full-length reads with an N50 length ≥ 1 kb, covering the complete transcript sequence.

[0082] 10. UMI + Internal Standard Combined Absolute Quantitative Analysis

[0083] 1. Raw data quality control: Filtering low-quality reads and removing connectors; 2. UMI clustering for deduplication: Using UMI+ alignment position as the unique identifier, PCR duplicates are removed to restore the original number of molecules; 3. Full-length transcript identification: Screening full-length reads to identify lncRNAs and their isoforms; 4. Internal standard quantification: Count the number of SIRV / ERCC reads and fit a standard curve (R²≥0.99). 5. Absolute quantitative correction: Corrects endogenous RNA reads based on a standard curve, outputting the true copy number in the copy number / ng level; 6. Isoform quantification: Differentiate different lncRNA isoforms and output the absolute expression level of each isoform.

[0084] In this embodiment, the above library construction and sequencing were performed using seven TSO-UMI fusion primers (Table 1) under the same conditions to verify their respective performance. The final results are shown in Table 3.

[0085] Table 3:

[0086] The above results are analyzed as follows: Sequence 1: Full length ratio 85%, UMI splitting efficiency 81%, CV=13%; Sequence 2: Amplification efficiency +9.86%, full length rate 87%, UMI resolution efficiency 81%, CV = 8.5%, polyA sequence capture efficiency +8.12%; Conclusion: Sequence 2 is optimal and is well-suited for high-precision quantification of lncRNA.

[0087] Example 2: Absolute Quantification of lncRNA in 293T Cells

[0088] I. RNA extraction from the sample to be tested

[0089] 1. Total RNA was extracted from 293T cells (5.0ug, RIN=8.7). 2. The specific execution process is the same as in Example 1; II. Sample rRNA removal (Ribo-off rRNA Depletion Kit (Human / Mouse / Rat)), the specific execution process is the same as in Example 1; 3. Use VAHTS RNA Clean Beads to purify Ribosomal-depleted RNA. The specific procedure is the same as in Example 1. IV. Reverse Transcription Adapter Ligation Two sets of reactions were set up using rRNA-Depletion-RNA, with two replicates for each set and the average of the results taken: Control group (CK1 / 2, prior patent original scheme): only 3' special connector + ordinary TSO, no UMI marking, no SIRV / ERCC internal standard; Experimental group (Exp1 / 2, complete technical solution of the present invention): TSO-UMI sequence 2 was selected as the reverse transcription primer and a complete SIRV-Set4 triple internal standard was incorporated.

[0090] V. The reverse transcription reaction generates cDNA, and the specific execution process is the same as in Example 1; VI. Amplification reaction, the specific execution procedure is the same as in Example 1; VII. Purify using 0.9X Ampure XP beads, following the same procedure as in Example 1; 8. Mix the PCR products, following the same procedure as in Example 1; 9. Use PCR mixed products to ligate sequencing adapters to construct libraries for sequencing. The specific execution process is the same as in Example 1. 10. UMI + internal standard combined absolute quantitative analysis, the specific execution process is the same as in Example 1.

[0091] In this embodiment, the library quality control data is shown in Table 4.

[0092] Table 4:

[0093] The lncRNA detection data are shown in Table 5-6.

[0094] Table 5:

[0095] Table 6:

[0096] Example 3: Quantification of lncRNA isoforms in mouse brain tissue

[0097] Sample: Mouse hippocampal RNA (500 ng, RIN=8.3); The process follows the steps in Example 1: rRNA removal → 3' adapter → TSO-UMI reverse transcription (sequence 2) → amplification → purification → ONT sequencing; Results: Full length percentage 89%, CV=8.8%; 12 lncRNA isoforms were accurately distinguished, with copy numbers ranging from 50 to 5000 copies / ng.

[0098] Example 4: Detection of trace lncRNAs in clinical liver cancer

[0099] Sample: Trace RNA from liver cancer tissue (500 ng, RIN=7.9); The process follows the steps in Example 1: rRNA removal → 3' adapter → TSO-UMI reverse transcription (sequence 2) → amplification → purification → ONT sequencing; Results: Low-abundance lncRNAs with ≥100 copies / ng were stably detected, with a CV ≤9.2%, which can serve as potential biomarkers.

[0100] The above results demonstrate that the library preparation and sequencing method provided by this invention successfully solves industry pain points such as PCR bias, relative quantification, batch differences, isoform identification, and quantification without polyA. The results prove that the full-length fraction is stable, the quantification is accurate, the isoform resolution is clear, and the method has a wide range of applications.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for absolute quantitative transcriptional library assembly of full-length lncRNA based on TSO-UMI labeling and compound internal standard correction, characterized by the following steps: include: S1. Extract total RNA from the sample and remove rRNA; S2. Add SIRV / ERCC composite internal standard; S3. Ligate the product from step S2 using reverse transcription adapter primers; S4. Add the TSO-UMI fusion primer and synthesize full-length cDNA by template switching; S5. Perform PCR amplification using cDNA as a template; S6. The amplified library was purified to obtain a full-length lncRNA absolute quantitative transcriptome library; The nucleotide sequence of the TSO-UMI fusion primer is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The method for absolute quantitative transcriptional assembly of full-length lncRNA as described in claim 1, characterized in that, The total RNA has a purity of A260 / A280 of 1.9-2.1 and an integrity of RIN ≥ 8.0; and / or the rRNA is removed to more than 95%.

3. The method for absolute quantitative transcriptional assembly of full-length lncRNA as described in claim 1, characterized in that, The composite internal standard was SIRV-Set 4 from Lexogen, added at an amount of 0.5-1% of total RNA.

4. The method for absolute quantitative transcriptional assembly of full-length lncRNA as described in claim 1, characterized in that, The reverse transcription adapter primers shown include primers with nucleotide sequences as shown in SEQ ID NO. 8-14.

5. The method for absolute quantitative transcriptional assembly of full-length lncRNA as described in claim 1, characterized in that, The PCR amplification process includes the addition of sequencing barcodes.

6. The method for absolute quantitative transcriptional assembly of full-length lncRNA as described in claim 1, characterized in that, The number of PCR amplification cycles is ≤13.

7. A full-length lncRNA absolute quantitative sequencing library, characterized in that, The database is obtained using the database construction method described in any one of claims 1-5.

8. A method for absolute quantitative transcriptome sequencing of full-length lncRNA, characterized in that, This includes loading the sequencing library described in claim 6 into an ONT sequencing chip, performing long-read sequencing to obtain sequences covering the complete transcript, filtering the sequences, and then performing lncRNA quantification analysis based on UMI and internal standards.

9. The sequencing method as described in claim 8, characterized in that, The quantitative analysis includes: a. Obtain the original number of molecules by removing duplicates using UMI clustering; b. Screen full-length reads to identify lncRNAs and their isoforms; c. Count the number of reads of the internal standard, correct the endogenous RNA according to the internal standard, and output the true copy number; d. Differentiate and output the absolute expression levels of each lncRNA isoform.

10. The sequencing method as described in claim 9, characterized in that, The calibration of the endogenous RNA was performed using the internal standard curve method.

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