Excess RNA depletion probes for high-value CDNA preparation with reverse transcription

Abstract

Provided are systems / kits and methods for the preparation of cDNA from a sample of total RNA, in which excess RNA species such as rRNA and tRNA are depleted from the cDNA products via prevention of reverse transcription through competitive binding of DNA oligonucleotide probes that prevent binding and / or extension of randomer reverse primers. This process allows downstream high-throughput sequencing on a library prepared from the cDNA to efficiently profile RNA expression without significant loss of accuracy due to excess RNA species occupying too many reads. Compared to other approaches for excess RNA depletion, such as those based on RNAse H systems, the systems and methods provided herein neither require additional enzymes beyond reverse transcriptase, nor extra purification before reverse transcription.

Description

Attorney Docket No. : 300402000940EXCESS RNA DEPLETION PROBES FOR HIGH-VALUE CDNA PREPARATION WITH REVERSE TRANSCRIPTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 679,268, filed August 5, 2024, which is incorporated herein by reference in its entirety.FIELD

[0002] The present disclosure relates generally to analysis of gene expression in biological RNA samples, and more specifically to reagents and methods for producing cDNA from an RNA sample that under-represents excess RNA species of known sequence.BACKGROUND

[0003] The analysis of gene expression in biological RNA samples is important for both life science research and biomedical diagnostics. RNA sequencing (RNAseq) is a high-throughput method for RNA expression analysis, and there are two primary methods for RNAseq.

[0004] Biological RNA samples can selectively be sequenced through either (1) reverse transcription using a polyT primer, or (2) mRNA enrichment using polyT probes functionalized on solid-phase separation beads. Such approaches allow all RNAseq reads to be applied to mRNA transcripts.However, such approaches are not applicable to degraded RNA samples, such as historical patient tissue samples stored in formalin-fixed, paraffin-embedded (FFPE) format. Furthermore, these approaches capture information about the roughly 30,000 mRNA species, but do not provide expression information on 300,000+ non-coding RNA (ncRNA) species.

[0005] Biological RNA samples can also be reverse transcribed using a polyN or other randomer DNA primer, most typically an N6 primer comprising a roughly equal mixture of all 4A6 = 4096 DNA hexamers. This approach is applicable to all RNA samples including degraded RNA samples, and provides expression information on ncRNA. However, without further methods, 90+% of the RNAseq would be wasted on a small number of excess RNA species, most notably ribosomal RNA (rRNA).

[0006] For the latter method, rRNA is typically depleted from the RNA sample. rRNA depletion is typically accomplished through the use of DNA probes complementary to the excess RNA and RNAse H. The rRNA depletion process is typically based on the selective degradation of RNA that is1MOFO-358193473Attorney Docket No. : 300402000940 hybridized to DNA. This approach is frequently used for total RNA sequencing that includes ncRNA, as well as for RNAseq of aged or damaged RNA samples. However, the rRNA depletion using RNAse H adds extra complexity to the workflow by requiring several additional steps, including both incubation with RNAse H and purification to remove RNAseq H. Furthermore, this approach is not compatible with high-throughput library preparation methods.BRIEF SUMMARY

[0007] In some aspects, provided is a system for producing cDNA from an RNA sample that underrepresents excess RNA species of known sequence, comprising: a set of oligonucleotide excess RNA depletion probes (ERD-Probes or “PERD”), wherein each ERD-Probe comprises a 5' sequence that is fully complementary to a subsequence of an excess RNA species and a 3' sequence or moiety that prevents extension by reverse transcription, and collectively the ERD-Probes hybridize to the excess RNA species in a fully-tiled manner, a DNA reverse transcription primer, a reverse transcriptase, and buffers and / or reagents for reverse transcriptase activity.

[0008] In one variations of the foregoing, the system is a kit for producing cDNA from an RNA sample that under-represents excess RNA species of known sequence.

[0009] Also provided are uses of such systems (or kits) described herein. In certain aspects, provided is a method for preparing cDNA from an RNA sample that under-represents excess RNA species of known sequence. In some embodiments, the method comprises: incubating the RNA sample and any of the systems described herein at a temperature amenable to reverse transcription.

[0010] In other embodiments, the method comprises: a) incubating a mixture comprising the RNA sample and a set of oligonucleotide excess RNA depletion probes (ERD-Probes) at a temperature so that the ERD-Probes hybridize to the excess RNA species in a fully-tiled manner, wherein each ERD-Probe comprises a 5' sequence that is fully complementary to a subsequence of an excess RNA species and 3' sequence or moiety that prevents extension by reverse transcription; b) adding to the mixture in step a) a DNA reverse transcription primer, a reverse transcriptase, and buffers and / or reagents for reverse transcriptase activity; and c) incubating the mixture of step b) at a temperature for reverse transcription.2MOFO-358193473Attorney Docket No. : 300402000940

[0011] In yet other embodiments, the method comprises: a) conducting thermal annealing on a mixture comprising the RNA sample with a set of oligonucleotide excess RNA depletion probes (ERD- Probes), wherein each ERD-Probe comprises a 5' sequence that is fully complementary to a subsequence of an excess RNA species and 3' sequence or moiety that prevents extension by reverse transcription; b) adding to the mixture in step a) DNA reverse transcription primer, a reverse transcriptase, and buffers and / or reagents for reverse transcriptase activity; and c) incubating the mixture of step b) at a temperature for reverse transcription.DESCRIPTION OF THE FIGURES

[0012] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0013] FIGURE 1: The RNA sample includes RNA species of interest and excess RNA species, both of which are potentially fragmented to varying extents. A set of Excess RNA Depletion Probes (ERD-Probes) is designed to bind the excess RNA species in a fully tiled manner, preventing binding and extension of a randomer reverse transcription primer.

[0014] FIGURE 2: Illustration of binding of the ERD-Probes to the fragmented excess RNA species. The vast majority of excess RNA species fragments are fully bound to ERD-Probes, with the exception of a small number of molecules wherein short 5' or 3' overhangs on the excess RNA species fragment are insufficiently long to bind to the ERD-Probes. Here, the ERD-Probes are designed to have a 3' sequence that does not hybridize to the excess RNA species. This feature of the ERD-Probes means that they will not be extended by a reverse transcriptase and act as a primer. Because many reverse transcriptase do not possess 3'->5' exonuclease activity, these 3' sequences will not be removed enzymatically. Because the modern synthesis of DNA oligonucleotides occurs from 3' to 5', the 3' nucleotides are unlikely to be missing due to oligonucleotide synthesis error.

[0015] FIGURE 3: Illustration of cDNA formation via reverse transcription of RNA of interest. RNA species of interest may exhibit partial non-specific binding to some ERD-Probes, but many consecutive nucleotides of the RNA species of interest will be single-stranded and available for binding the polyN reverse transcription primer. Because the reverse transcriptase possesses strand displacement activity, the extending primer will be uninhibited by the potential nonspecific binding of ERD-Probes to RNA species of interest.3MOFO-358193473Attorney Docket No. : 300402000940

[0016] FIGURE 4: Illustration of variants of the ERD-Probe structure and the reverse transcription primer structure. In some embodiments, ERD-Probes comprise uracil (U) nucleotides at one or more positions in replacement of thymine (T) nucleotides. These embodiments allow enzymatic degradation of the ERD-Probes using UDG or USER enzymes after reverse transcription. In some embodiments, the ERD-Probe comprises a 3' chemical modification that prevents reverse transcriptase extension, such as a 3-carbon spacer (C3), a phosphate, and inverted nucleotide. These embodiments prevent potential undesirable reverse transcription using ERD-Probes as a primer that nonspecifically bind to other ERD- Probes. In some embodiments, an additional ERD-Protector oligonucleotide partially complementary to each ERD-Probe is introduced at the same time as the ERD-Probes for excess RNA depletion. These embodiments reduce the nonspecific binding of ERD-Probes to RNA species of interest with significant sequence homology to the excess RNA species. In some embodiments, the reverse transcription primer comprises a hairpin structure to the 5' of a randomer (e.g. N6) primer sequence. These embodiments allow the primer to preferentially bind to the 3' end of RNA of interest, increase the fraction of cDNA and NGS reads that are longer and can be more easily aligned to the transcrip tome. In some embodiments, the reverse transcription primer is a mixamer comprising a number of repeats of (S / WW), comprising a strong (S) cytosine or guanine nucleotide or two weak (W) adenine or thymine nucleotides. These embodiments allow more unbiased binding of the reverse transcription primer to various different RNA sequences. In some embodiments, the reverse transcription primer is not a randomer sequence such as N_6, but rather a set of gene-specific reverse transcription primer sequences. These embodiments allow higher sensitivity and representation in the cDNA of the RNA species specifically targeted by the reverse transcription primers. The variants described, as well as other variants, could be implemented in any suitable combinations, and are not mutually exclusive with one another.

[0017] FIGURE 5: Illustration of a variant ERD-Probe with an ancillary protector oligonucleotide that is partially complementary to every ERD-Probe. The protector oligonucleotide outcompetes the RNA of interest in binding to the ERD-Probe oligonucleotides, but is outcompeted by the excess RNA sequences that hybridize to the ERD-Probe oligonucleotides with more base pairs.

[0018] FIGURE 6: Illustration of a variant reverse transcription primer with a hairpin structure to the 5' of an N6 priming region. These hairpin polyN reverse transcription primers will preferentially bind to the 3' end of RNA species of interest, because binding at the 3' end results in an extra thermodynamically stabilizing base stack between the 3' end base pair of the RNA species of interest and the first base pair of the hairpin.4MOFO-358193473Attorney Docket No. : 300402000940

[0019] FIGURE 7 : One possible workflow for use of ERD-Probes in constructing a cDNA mixture from an RNA mixture that is under-represented in the excess RNA species. In this embodiment, the ERD-Probes are first added to the RNA sample, allowed to incubate or thermally anneal with the RNA sample, and then the reverse transcription (RT) primer and reverse transcriptase are subsequently added to the mixture. Then reverse transcription proceeds, resulting in a mixture of RNA with cDNA, in which the cDNA is under-represented in the excess RNA species due to the inhibitory effects of the ERD- Probes.

[0020] FIGURE 8: Illustration of variant ERD-Probe with a UV-crosslinking moiety such as psoralen or CNVK. DNA oligonucleotides functionalized with CNVK will covalently bind to the hybridized nucleic acid molecule upon exposure to ultraviolet light. This property can be advantageous for improving the suppression of the excess RNA. If one or a few ERD-Probes have low yield in binding to its excess RNA target, such as because the ERD-Probe has significantly secondary structure or because it has low G / C content, the crosslinking of the other ERD-Probes that bind to the same excess RNA molecule will prevent the reverse transcription primer from being extended completely.

[0021] FIGURE 9: Differential expression analysis of Rnl8s (ERD probe target gene) and other top 1000 genes in mouse whole blood. Comparison of expression values (reads per million total reads, RPM) of Rnl8s and the other top 1000 most expressed genes between the control group (without ERD) and the experimental group (with ERD). Expression of Rnl8S decreased from 38.02% (without ERD) to 0.16% (with ERD), demonstrating the significant impact of ERD treatment on Rnl8s expression.

[0022] FIGURE 10: Concordance analysis of gene expression with and without ERD: Orthogonal plot of the log2 scale RPM values for the top 1000 most expressed genes (excluding Rnl8s) in the control group (without ERD) versus the experimental group (with ERD). This plot shows good concordance of gene expression levels between the two conditions, indicating the high specificity of ERD probes.

[0023] FIGURE 11: PERD Technology Performance Metrics. (A) Depletion efficiency across species (human, mouse, rat), demonstrating consistent high-efficiency rRNA removal (up to 99.8%) regardless of species origin. (B) Nucleotide coverage profile of depleted rRNA, illustrating uniform depletion across the entire length of ribosomal RNA transcripts without positional biases. The gray lines and black lines indicate the nucleotide coverage of ribosomal RNA transcripts in each species from total5MOFO-358193473Attorney Docket No. : 300402000940RNAseq and BIRT+PERD data, respectively. The percentage values in brackets indicate the reads alignment rate to associated ribosomal RNA transcripts.DETAILED DESCRIPTION

[0024] The following description sets forth exemplary compositions, systems / kits, methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.Excess RNA Depletion Probe (ERD-Probe or “PERD”) Approach

[0025] In some aspects, provided here are probes for excess RNA depletion (referred to herein as ERD-Probe or “PERD”). This is an enzyme-free method for removing ribosomal RNA, which typically makes up around 90% of cellular RNA but provides little useful information. Traditional methods use expensive enzymes like RNase H, but the systems and methods herein use probes that selectively deplete rRNA without additional enzymatic steps, further reducing costs while maintaining high data quality. Together, such systems and methods reduce per-sample costs significantly compared to traditional methods, making large-scale RNA sequencing economically feasible.

[0026] In some aspects, provided is a method that uses DNA hybridization probes that bind to excess RNA species in a fully-tiled manner, leaving no binding sites for polyN or other randomer reverse primers to initiate reverse transcription. The DNA hybridization probes comprise a 3' sequence or modification that prevents extension by reverse transcription, preventing the ERD-Probes from acting as reverse transcription primers. All RNA species other than the excess RNA species targeted by the ERD- Probes may exhibit a minor degree of nonspecific binding to ERD-Probes, but are expected to possess sufficiently many unpaired nucleotides even after nonspecific ERD-Probe binding that allows the DNA reverse transcription primer to bind and extend.

[0027] Mathematically, it is possible to define the expression (concentration) of excess RNA species in the RNA sample as [ER]_i, where i is the index of the excess RNA species, and the expression (concentration) of all other RNA species (also known as RNA of interest) as [ROI] _j, where j is the index of the RNA species of interest. During standard reverse transcription by a N_6 DNA randomer reverse primer to form cDNA, there will be some degree of bias b_i and b J for each ER and ROI.Thus, the cDNA concentrations of ER and ROI can be represented as, as shown in Eqs. 1 and 2:6MOFO-358193473Attorney Docket No. : 300402000940[c_ER]_i = [ER]_i * b_i Eq. 1[c_ROI] J = [ROI] J * bj Eq. 2

[0028] Through the process of excess RNA depletion, either by ERD-Probes or by traditional RNAse H-based approaches, the concentration of the cDNA becomes, as shown in Eqs. 3 and 4:[c_ER]_i = [ER]_i * b_i * (1 - Y_i) Eq. 3[c_ROI] J = [ROI] J * bj * dj Eq. 4 where Y_i is the depletion yield of ER_i, and d j is the differential yield of ROI J. In some embodiments, d_j is normalized so that Mean(d_j) = 1. An ideal excess RNA depletion approach will maximize Y_i while minimizing the standard deviation of d_j. In some embodiments, Mean(Y_i) > 0.98 and Std(d_j) < 0.1 are desirable.

[0029] The architecture, sequence design, and concentrations of the ERD-Probes impact the systemlevel Mean(Y_i) and Std(dJ) metrics via two lower-level properties: the binding yield of ERD-Probes to ER species, and the binding yield of ERD-Probes to ROI species. ERD-Probes' sequences are designed to be complementary to ER species, in a way that collectively fully tile the ER species. In contrast, except in the case of ROI with sequence highly homologous to that of an ER, the ROI are mismatched to the ERD-Probes, and will bind with both lower thermodynamic stability and slower kinetics. Generally speaking, ERD-Probes that are longer and more G / C rich will bind with stronger thermodynamics, quantitated as the Gibbs standard free energy of hybridization to the corresponding complementary ER sequence (AG°). Longer and / or more G / C rich ERD-Probes with stronger (more negative) AG° also generally tend to bind with faster kinetics, though this correlation is imperfect due to the potential formation of secondary structures in the ERD-Probes and the corresponding ER subsequences that can dramatically slow the hybridization reaction.

[0030] ERD-Probes with extremely strong AG° will also generally tend to have very strong nonspecific binding to unintended ROI species, consequently ERD-Probes with an intermediate AG° at the hybridization temperature and salinity may strike a good balance between high Mean(Y_i) and low Std(d_j). In some embodiments, all of the ERD-Probes are designed to that their AG° are all within a range of a target value AG°_target at a predefined temperature and salinity. In other embodiments, the hybridization temperature and salinity are selected for a pre-defined set of ERD-Probes to achieve a7MOFO-358193473Attorney Docket No. : 300402000940 desired range of AG° values. In other embodiments, the ERD-Probes have nonbinding sequences at their 5' or 3' regions not complementary to their corresponding ERs, in order to achieve uniform ERD-Probe length and roughly uniform AG° values. For example, a G / C poor ERD-Probe of length 60 nucleotides may be designed with another G / C rich ERD-Probe with binding region of only 25 nucleotides and 35 additional "padding" nucleotides that serve only to make the probes uniformly 60nt for considerations such as ease of synthesis or ease of purification.

[0031] In some embodiments, the ERD-Probes are introduced to the RNA sample at the same time as the DNA reverse transcriptase (RT) primer at an elevated temperature where the ERD-Probes can stably hybridize ER species but the DNA RT primer cannot stably hybridize. After allowing ERD-Probe hybridization at the elevated temperature for a period of time, the temperature is lowered to allow the DNA RT primer to bind to ROI species. In other embodiments, the ERD-Probes are introduced to the RNA sample first, and allowed time to hybridize to the ER species. After allowing the ERD-Probe hybridization, the DNA RT primers are then added to the reaction mixture to allow binding to unhybridized regions of the ROI species.

[0032] In some embodiments, the DNA RT primers are a standard N_6 primer, in which all four nucleotides (A, T, C, G) are introduced at each position with roughly equal concentration during each of the 5 nucleotide addition reactions, resulting in a roughly equal mixture of all 4A6 hexamer species.This N_6 DNA RT primer can, in principle, bind to every 6nt subsequence of each ROI that is not nonspecifically hybridized to an ERD-Probe. Because the reverse transcriptase possesses strand displacement activity, the N_6 primer can be extended and produce long cDNA despite ERD-Probes binding nonspecifically to the same ROI molecule as the N_6 primer. However, significant ERD-Probe binding to a specific ROI species will reduce the number of continuous 6nt unpaired subsequences available, resulting in under-representation of that ROI (lower d_j). Furthermore, although reverse transcriptase possesses strand displacement activity, enzymatic extension of the primer is generally believed to be slower when strand displacement needs to occur, also leading to lowered d_j . Consequently, strong ERD-Probe binding to ROI species may not be desirable.

[0033] In some embodiments, the DNA RT primer comprises a hairpin structure to the 5' of the N_6 sequence at the 3' end. The hairpin structure can help thermodynamically stabilize the binding of the DNA RT primer to the 3' end of an ROI molecule, relative to the middle of an ROI molecule. This is because binding the hairpin primer to the 3' end results in an extra DNA base stack between the hairpin8MOFO-358193473Attorney Docket No. : 300402000940 stem and the 3 '-most base pair of the ROI. In contrast, the hairpin primer binding to the middle of an ROI molecule would result in a thermodynamically destabilizing dangle from the 3' end of the ROI. This effect is well-documented in literature, and is particularly why foldback primers with hairpin structures are used to amplify microRNA sequences in PCR. Other variants of ERD-Probes and DNA RT primers can also be constructed to improve ERD-Probe binding specificity or uniformity, as illustrated in FIGURES 4-8.

[0034] In some embodiments, the ERD-Probes comprise uracil (U) nucleotides at positions replacing thymine (T) nucleotides, in order to facilitate downstream degradation and removal by size selection. The UDG enzyme and the USER enzyme mix selectively cleaves DNA at U bases. In some embodiments, after the DNA RT primer has been extended by reverse transcriptase to form cDNA, and then the reverse transcriptase has been thermally or chemically inactivated, UDG or USER enzymes are added to degrade the ERD-Probes. In some embodiments, the degraded ERD-Probes are removed through size selection, such as via SPRI beads.

[0035] In some embodiments the ERD-Probes are designed to deplete human, mouse and rat cytoplasmic ribosomal RNAs for the 5S (Rn5s), 5.8S (Rn5.8s), 18S (Rnl8s), and 28S (Rn28s) subunits. In other embodiments, the ERD-Probes additionally deplete cytoplasmic rRNA for the internal transcribed spacer (ITS) and external transcribed spacer (ETS).

[0036] In some embodiments the ERD-Probes are designed to deplete human, mouse and rat mitochondrial ribosomal RNAs for the 12S (mt-RNRl) and 18S (mt-RNR2) subunits.

[0037] In some embodiments the ERD-Probes are designed to deplete human, mouse and rat embryonic and fetal globin mRNAs (HBA1, HBA2, HBB, HBD, HBM, HBG1, HBG2, HBE1, HBQ1 and HBZ).

[0038] In one aspect, provided is system for producing cDNA from an RNA sample that underrepresents excess RNA species of known sequence, comprising: a set of oligonucleotide excess RNA depletion probes (ERD-Probes), wherein each ERD-Probe comprises a 5' sequence that is fully complementary to a subsequence of an excess RNA species and 3' sequence or moiety that prevents extension by reverse transcription, and collectively the ERD-Probes hybridize to the excess RNA species in a fully-tiled manner, a DNA reverse transcription primer,9MOFO-358193473Attorney Docket No. : 300402000940 a reverse transcriptase, and buffers and / or reagents for reverse transcriptase activity.

[0039] In some embodiments, the ERD-Probes are DNA. In some variations, the 3' sequence of a ERD-Probe comprise between 2 and 10 nucleotides with sequences that are not complementary to the corresponding 5' sequence of the excess RNA species that the 5' sequence of the ERD-Probe is complementary to. In some variations, the 3' moiety of a ERD-Probe comprise a phosphate, a 3-carbon- spacer, an inverted nucleotide, a biotin, an azide, or an alkyl group. In some embodiments, the ERD- Probes deplete a same target gene from a plurality of different organisms. In certain embodiments, the ERD-Probes fully tile the target gene from each organism in the plurality of different organisms. In some other embodiments, highly conserved regions for the target gene among the plurality of different organisms share a same set of ERD-Probes, and wherein nonhomologous regions of the target gene among the plurality of different organisms have a unique set of ERD-Probes for each organism in the plurality of different organisms. In some embodiments, the same set of ERD-Probes fully tile the target gene for at least one organism in the plurality of organisms, partially tile the target gene for remaining organisms in the plurality of different organisms, and wherein the unique set of ERD-Probes for each organism tile the remainder of the target gene for the remaining organisms in the plurality of different organisms.

[0040] In certain variations, each ERD-Probe independently comprises between 20 and 60 nucleotides. In certain variations, the ERD-Probes hybridize to their corresponding excess RNA species with a Gibbs standard free energy of between - 15 kcal / mol and -50 kcal / mol at a temperature of 20 °C and 65 °C in an equivalent salinity of 1 Molar sodium ions.

[0041] In some embodiments, the DNA reverse transcription primer comprises a randomer sequence comprising degenerate nucleotides. In some variations, the DNA reverse transcription primer comprises 6 consecutive N nucleotides, wherein each N is a roughly equal mixture of guanine (G), cytosine (C), adenine (A), and thymine (T). In certain variations, the DNA randomer reverse transcription primer comprises 5 consecutive (S / WW) groups of nucleotides, wherein each (S / WW) is a mixture of guanine (G), cytosine (C), adenine-adenine (AA), adenine-thymine (AT), thymine-adenine (TA), and thyminethymine (TT). In certain variations, the DNA randomer reverse transcription primer further comprises a 5' sequence with a hairpin structure. In one variation, the hairpin structure has a stem length of between10MOFO-358193473Attorney Docket No. : 3004020009406 and 20 base pairs, and a loop length of between 3 and 10 nucleotides. In a certain variation, the loop has a sequence that contains a sample-specific barcode sequence.

[0042] In other embodiments, the DNA reverse transcription primer comprises a plurality of genespecific DNA oligonucleotide primers, with sequence complementary to subsequences of the excess RNA species.

[0043] In some variations, the ERD-Probes further comprise one or more uracil (U) nucleotides in place of thymine (T) at positions complementary to adenine (A) on the corresponding excess RNA species.

[0044] In other embodiments, the system further comprising a set of oligonucleotide excess RNA depletion protectors (ERD-Protectors), wherein each ERD-Protector is partially complementary to each ERD-Probe.

[0045] In some aspects, provided is a method for preparing cDNA from an RNA sample that underrepresents excess RNA species of known sequence, the method comprising: incubating the RNA sample and any of the aforementioned embodiments and variations of the system described herein at a temperature amenable to reverse transcription.

[0046] In certain aspects, provided is a method for preparing cDNA from an RNA sample that under-represents excess RNA species of known sequence, the method comprising: a) incubating a mixture comprising the RNA sample and a set of oligonucleotide excess RNA depletion probes (ERD-Probes) at a temperature so that the ERD-Probes hybridize to the excess RNA species in a fully-tiled manner, wherein each ERD-Probe comprises a 5' sequence that is fully complementary to a subsequence of an excess RNA species and 3' sequence or moiety that prevents extension by reverse transcription; b) adding to the mixture in step a) a DNA reverse transcription primer, a reverse transcriptase, and buffers and / or reagents for reverse transcriptase activity; and c) incubating the mixture of step b) at a temperature for reverse transcription.

[0047] In certain aspects, provided is a method for preparing cDNA from an RNA sample that under-represents excess RNA species of known sequence, the method comprising:11MOFO-358193473Attorney Docket No. : 300402000940 a) conducting thermal annealing on a mixture comprising the RNA sample with a set of oligonucleotide excess RNA depletion probes (ERD-Probes), wherein each ERD-Probe comprises a 5' sequence that is fully complementary to a subsequence of an excess RNA species and 3' sequence or moiety that prevents extension by reverse transcription; b) adding to the mixture in step a) DNA reverse transcription primer, a reverse transcriptase, and buffers and / or reagents for reverse transcriptase activity; and c) incubating the mixture of step b) at a temperature for reverse transcription.

[0048] In some embodiments, the thermal annealing comprises lowering the temperature of the mixture from not lower than 75 °C to not higher than 45 °C. In certain other embodiments, the thermal annealing comprises lowering the temperature of the mixture from not higher than 96 °C to not lower than 45 °C. In some variations, the temperature of the mixture is lowered at a speed of not faster than 1 °C / second.

[0049] In some other embodiments, the thermal annealing comprises heating the mixture at a temperature between 45 °C and 75 °C. In certain other embodiments, the thermal annealing comprises heating the mixture at a temperature between 45 °C and 96 °C. In some variations, the temperature of the mixture is lowered at a speed of not faster than 1 °C / second.

[0050] In some variations, at least one reagent for reverse transcriptase activity is one or more dNTPs.

[0051] In some embodiments, “stem length” in a hairpin structure is the length of the doublestranded segment (stem) where nucleotide bases pair through hydrogen bonds.

[0052] In some embodiments, “loop length” in a hairpin structure is the number of bases within the loop of the hairpin structure that are unpaired.Advantages of ERD-Probe Approach for Excess RNA Depletion

[0053] One method for excess RNA depletion is through use of RNAse H. In such a method, DNA probes are used to hybridize to excess RNA species. Subsequently, RNAse H is used to cleave RNA bound to these DNA probes. Because the probes preferentially bind ER species rather than ROI species, RNAse H also achieves depletion of ER in the cDNA products. The disadvantage of this approach is first that probes nonspecifically bound to ROI will also induce cleavage of the ROI species, resulting in 12MOFO-358193473Attorney Docket No. : 300402000940 sequencing bias and lowered d j for some species, increasing over Std(dJ). Second, this requires an extra enzyme RNAse H with a different set of required buffer conditions than reverse transcriptase. The RNAse H and its corresponding reaction buffer must be removed by purification before the reverse transcriptase is introduced and used to create cDNA, adding labor and time to the process.

[0054] There are a few significant differences between the ERD-Probes presented here and the probes used for RNAse H treatment: First, ERD-Probes have a 3' sequence or modification that prevents reverse transcriptase extension. In contrast, because the probes used for RNAse H are removed before reverse transcription, probes for RNAse H do not require the 3' nonextensible sequence or moiety. Second, ERD-Probes are designed to exactly tile the ER species. This is because imperfect coverage of the ER species would result in remaining sites for DNA reverse transcription primers to bind, and because redundant coverage of ER species would result in competitive hybridization by multiple probes that can result in probe reverse transcriptase extension or imperfect coverage of ER species.

[0055] Thus, in some embodiments, the systems and methods herein do not use RNAse H. In some variations, the systems and methods herein selectively deplete rRNA without additional enzymatic steps. This helps to reduce costs while maintaining high data quality.

[0056] In some embodiments, it is desirable to deplete transcripts for versions of a gene that occur across multiple species. The implementation of the optimal PERD primer mixture would depend on the relatedness of the species and the homology between the depleted genes of interest. For example, a commercial kit containing PERD primers might provide PERD primers for deleting the Rnl8s gene in three distantly related species, e.g. human, octopus, and drosophila, so that a single kit can be purchased to cover all three species. In some embodiments, PERD primers are designed so that the mix of primers contains unique PERD primers tiling the entire human Rnl8s gene, the entire octopus Rnl8s gene, and the entire drosophila Rnl8s gene. Given that the Rnl8s gene in all three species requires N primers for tiling, the mixture contains 3 times N primers, to cover all three versions of the gene.

[0057] In certain other embodiments for which the species of interest are more closely related, and the genes to be depleted are more closely related, a more economical and efficient approach to designing PERD primers is used. For example, a commercial kit containing PERD primers might provide PERD primers for deleting the Rnl8s gene in three closely related species, human, mouse, and rat. In such an embodiment, one species would be designated the "base species" and the mix of primers would be13MOFO-358193473Attorney Docket No. : 300402000940 designed so that it includes PERD primers tiling the entire Rnl8s gene for the base species (e.g. mouse). Further, the other two species would be designated "add-on" species (e.g. human and rat) and primers corresponding to these two species Rnl8s genes would be added only for those regions of the human and rat Rnl8s genes for which there was low homology with the mouse Rnl8s gene. In this case the total number of PERD primers for depleting all three species would be only N + x + y, where x and y represent a small number of primers which cover regions of the Rnl8s gene for which the human (x) and rat (y) differ significantly from the mouse Rnl8s gene.

[0058] In some variations, as described below in example 2, ERD probe sequences were designed that target mouse, human, and rat species in an effort to make the methods for tiling described herein universally applicable. First, efforts were made to fully tile the mouse rRNA sequence. Then, the rRNA sequences of human and rat were analyzed and found to share over 90% sequence similarity with mouse rRNA. For the remaining 10% of non-conserved rRNA regions, additional probes were designed to fully tile these unique segments in human and rat. In this example, one only needs to tile the species-specific 10% of the human and rat rRNA sequences. In this regard, these sequences may be referred to as being “partially tiled.” Because the remaining 90% of the sequence is identical to the mouse sequence, the existing mouse-targeting probes can also hybridize with human and rat rRNA. Therefore, the final probe set effectively achieves full tiling coverage across all three species.EXAMPLES

[0059] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.Example 1: ERD Probe Design and UseERD Probe Design

[0060] The ERD probes were designed with the following specifications:1. 3' Phosphate Modifications: To prevent extension during cDNA synthesis.2. Tiling through C57BL / 6 Mouse 18S rRNA Sequence: The probes were designed to tile across the 18S rRNA sequence of the C57BL6 mouse strain.3. Thermodynamic Stability: The probes were designed to have a binding free energy (AG) of around -20 kcal / mol.14MOFO-358193473Attorney Docket No. : 300402000940Experiment WorkflowRNA Extraction from Mice Whole Blood Samples

[0061] For RNA extraction from mice whole blood, 100 pL of the sample was mixed with 300 pL of Tri-zol. RNA extraction was performed using commercially available kits. Quality control (QC) of the extracted RNA was conducted using commercially available kits, methods / techniques and equipment (e.g., bioanalyzer).Experiment Design

[0062] Two groups were designed for the experiment:• Group 1: Control Group - Extracted total RNA directly proceeded to the cDNA synthesis workflow.• Group 2: Experiment Group - Extracted total RNA was mixed with the ERD probe, followed by a heat and anneal process, and then proceeded directly to the downstream cDNA synthesis workflow. cDNA Synthesis

[0063] For both groups, cDNA synthesis was carried out as follows: First strand cDNA synthesis was performed, followed by second strand cDNA synthesis. cDNA purification was then conducted. The foregoing steps were performed using commercially available kits and methods / techniques.Library Preparation

[0064] Library preparation for synthesized double-stranded cDNA was performed. The cDNA is ligated with adaptors, and a qPCR test was conducted to determine the amplification cycles for adaptor- ligated cDNA. The final library, with dual unique barcodes, was generated. The foregoing steps were performed using commercially available kits and methods / techniques.Library QC15MOFO-358193473Attorney Docket No. : 300402000940

[0065] QC for the final library was performed using commercially available kits, methods / techniques and equipment (e.g., bioanalyzer).Library Sequencing

[0066] Sequencing of QC-passed libraries was performed using 2 x 150 paired-end sequencing on commercially available equipment.Bioinformatic Analysis

[0067] The sequencer generated FASTQ files, which were initially trimmed to remove certain adaptor sequences. Subsequently, alignment with the C57BL / 6 Mouse reference genome was performed, generating alignment files. Gene expression quantification was performed. Normalization by the total number of reads of each library is performed before gene expression comparison analysis.Results

[0068] Table 1 below summarizes the comparison of Rnl8s expression in control group and experiment group. See also FIGURES 9 and 10.Table 1. Comparison of Rnl8s Expression with ERD probes and without ERD probeExample 2: Cross-Species ERD Probe Design with Thermodynamically Optimized, Nucleotide- Resolution DepletionERD Probe Design

[0069] ERD probes were designed to achieve high-efficiency depletion of ribosomal RNA (rRNA) across three mammalian species: Mus musculus (C57BL / 6 mouse), Rattus norvegicus (Sprague-Dawley rat), and Homo sapiens (human). The probe design strategy included:16MOFO-358193473Attorney Docket No. : 3004020009401. Multi-Species Homology-Based Tiling:

[0070] Multiple sequence alignment of 18S and 28S rRNA sequences from the three species was performed to identify highly conserved regions. ERD probes were designed to tile across these homologous regions to enable broad and efficient depletion across all target species.2. Species-Specific Region Tiling:

[0071] Additional probes were designed to cover unique or divergent regions within the 18S and 28S rRNA of each individual species, ensuring complete coverage of species-specific sequences that are not captured by conserved tiling.3. Depletion-Guided Probe Augmentation:

[0072] After an initial round of ERD probe validation, RNA-seq coverage profiles were analyzed to identify nucleotide regions with suboptimal depletion. A supplementary set of probes was then designed to target these residual signal regions. This adaptive strategy enabled uniform and nucleotide-level depletion of the entire rRNA transcript.4. Thermodynamic Optimization:

[0073] All probes were engineered to have a hybridization free energy (AG) of approximately -35 kcal / mol, enabling the use of longer probes for improved binding affinity and thermodynamic stability. Each probe also incorporated a 3’ phosphate modification to prevent extension during reverse transcription.Experimental Workflow

[0074] Total RNA was extracted from whole blood of mouse, rat, and human using Tri-zol reagent followed by silica membrane purification. RNA integrity was assessed via capillary electrophoresis using a bioanalyzer or equivalent instrument.Experimental Design

[0075] For each species, samples were split into two groups:17MOFO-358193473Attorney Docket No. : 300402000940• Control Group: RNA was directly processed through the cDNA synthesis workflow without ERD probe treatment.• ERD Group: RNA was incubated with the ERD probe mixture, subjected to a denaturation and annealing protocol, and then advanced to cDNA synthesis.Library Preparation and Sequencing

[0076] All samples were processed through identical workflows involving first- and second-strand cDNA synthesis, adaptor ligation, indexing, and PCR amplification using commercially available kits. Final libraries were quality-checked and sequenced with paired-end 2x 150 bp reads on Illumina instruments.Bioinformatics Analysis

[0077] Sequencing data were trimmed and aligned to the appropriate species reference genome. Reads mapping to the 18S and 28S rRNA regions were quantified, and depletion efficiency was calculated at single-nucleotide resolution.Results

[0078] As shown in Figure 11 (inset A), the total number of reads mapping to rRNA sequences was reduced by more than 99.8% in all three species following ERD treatment. Importantly, Figure 11 (inset B) demonstrates the uniform and continuous depletion across the entire 18S and 28S transcripts. The blue-shaded regions indicate the designed probe coverage, and the depletion patterns closely match probe locations. Regions marked with red brackets represent previously low-depletion zones, which were successfully targeted and depleted after the second round of probe augmentation. This confirms that the ERD strategy achieved nucleotide-level depletion of all target rRNA sequences, including hard- to-deplete areas.

[0079] This example illustrates that a thermodynamically optimized, tiered probe design — consisting of conserved, species-specific, and depletion-guided probes — can achieve near-complete depletion of rRNA across multiple mammalian species. The use of longer probes with AG ~ -35 kcal / mol, combined with iterative design refinement, enables nucleotide-resolution depletion of 18S and 28S rRNA, as18MOFO-358193473Attorney Docket No. : 300402000940 demonstrated in Figure 11. This approach substantially enhances sequencing efficiency and transcriptome coverage.19MOFO-358193473

Claims

Attorney Docket No. : 300402000940CLAIMSWhat is claimed is:

1. A system for producing cDNA from an RNA sample that under-represents excess RNA species of known sequence, comprising: a set of oligonucleotide excess RNA depletion probes (ERD-Probes), wherein each ERD-Probe comprises a 5' sequence that is fully complementary to a subsequence of an excess RNA species and a 3' sequence or moiety that prevents extension by reverse transcription, and wherein, collectively the ERD-Probes hybridize to the excess RNA species in a fully-tiled manner, a DNA reverse transcription primer, a reverse transcriptase, and buffers and / or reagents for reverse transcriptase activity.2 The system of claim 1 , wherein the ERD-Probes are DNA.3 The system of claim 1 or 2, wherein the 3' sequence of an ERD-Probe comprises between 2 and 10 nucleotides with sequences that are not complementary to the corresponding 5' sequence of the excess RNA species that the 5' sequence of the ERD-Probe is complementary to.4 The system of claim 1 or 2, wherein the 3' moiety of an ERD-Probe comprises a phosphate, a 3- carbon-spacer, an inverted nucleotide, a biotin, an azide, or an alkyl group.5 The system of any one of claims 1 to 4, wherein the ERD-Probes deplete a same target gene from a plurality of different organisms.6 The system of claim 5, wherein highly conserved regions for the target gene among the plurality of different organisms share a same set of ERD-Probes, and wherein nonhomologous regions of the target gene among the plurality of different organisms have a unique set of ERD-Probes for each organism in the plurality of different organisms.20 OFO-358193473Attorney Docket No. : 3004020009407. The system of claim 5, wherein the ERD-Probes fully tile the target gene from each organism in the plurality of different organisms.

8. The system of claim 6, wherein the same set of ERD-Probes fully tile the target gene for at least one organism in the plurality of organisms, partially tile the target gene for remaining organisms in the plurality of different organisms, and wherein the unique set of ERD-Probes for each organism tile the remainder of the target gene for the remaining organisms in the plurality of different organisms.

9. The system of any one of claims 1 to 8, wherein each ERD-Probe independently comprises between 20 and 60 nucleotides.

10. The system of any one of claims 1 to 8, wherein the ERD-Probes hybridize to their corresponding excess RNA species with a Gibbs standard free energy of between -15 kcal / mol and -50 kcal / mol at a temperature between 20 °C and 65 °C in an equivalent salinity of 1 Molar sodium ions.

11. The system of any one of claims 1 to 10, wherein the DNA reverse transcription primer comprises a randomer sequence comprising degenerate nucleotides.

12. The system of any one of claims 1 to 11, wherein the DNA reverse transcription primer comprises 6 consecutive N nucleotides, wherein each N is a roughly equal mixture of guanine (G), cytosine (C), adenine (A), and thymine (T).

13. The system of any one of claims 1 to 12, wherein the DNA reverse transcription primer comprises 5 consecutive (S / WW) groups of nucleotides, wherein each (S / WW) is a mixture of guanine (G), cytosine (C), adenine-adenine (AA), adenine-thymine (AT), thymine-adenine (TA), and thyminethymine (TT).

14. The system of any one of claims 1 to 13, wherein the DNA reverse transcription primer further comprises a 5' sequence with a hairpin structure.

15. The system of claim 14, wherein the hairpin structure has a stem length of between 6 and 20 base pairs, and a loop length of between 3 and 10 nucleotides.

16. The system of claim 15, wherein the loop has a sequence that contains a sample- specific barcode sequence.21MOFO-358193473Attorney Docket No. : 30040200094017. The system of any one of claims 1 to 10, wherein the DNA reverse transcription primer comprises a plurality of gene-specific DNA oligonucleotide primers, with a sequence complementary to subsequences of the excess RNA species.

18. The system of any one of claims 1 to 17, wherein the ERD-Probes further comprise one or more racil (U) nucleotides in place of thymine (T) at positions complementary to adenine (A) on the corresponding excess RNA species.

19. The system of any one of claims 1 to 18, further comprising a set of oligonucleotide excess RNA depletion protectors (ERD-Protectors), wherein each ERD-Protector is partially complementary to each ERD-Probe.

20. A method for preparing cDNA from an RNA sample that under-represents excess RNA species of known sequence, the method comprising: incubating the RNA sample and the system of any one of claims 1 to 19 at a temperature amenable to reverse transcription.

21. A method for preparing cDNA from an RNA sample that under-represents excess RNA species of known sequence, the method comprising: a) incubating a mixture comprising the RNA sample and a set of oligonucleotide excess RNA depletion probes (ERD-Probes) at a temperature so that the ERD-Probes hybridize to the excess RNA species in a fully-tiled manner, wherein each ERD-Probe comprises a 5' sequence that is fully complementary to a subsequence of an excess RNA species and a 3' sequence or moiety that prevents extension by reverse transcription; b) adding to the mixture in step a) a DNA reverse transcription primer, a reverse transcriptase, and buffers and / or reagents for reverse transcriptase activity; and c) incubating the mixture of step b) at a temperature for reverse transcription.

22. A method for preparing cDNA from an RNA sample that under-represents excess RNA species of known sequence, the method comprising: a) conducting thermal annealing on a mixture comprising the RNA sample with a set of oligonucleotide excess RNA depletion probes (ERD-Probes), wherein each ERD-Probe comprises a 5'22 OFO-358193473Attorney Docket No. : 300402000940 sequence that is fully complementary to a subsequence of an excess RNA species and a 3' sequence or moiety that prevents extension by reverse transcription; b) adding to the mixture in step a) DNA reverse transcription primer, a reverse transcriptase, and buffers and / or reagents for reverse transcriptase activity; and c) incubating the mixture of step b) at a temperature for reverse transcription.

23. The method of claim 22, wherein the thermal annealing comprises lowering the temperature of the mixture from not higher than 96 °C to not lower than 45 °C.

24. The method of claim 23, wherein the temperature of the mixture is lowered at a speed of not faster than 1 °C / second.

25. The method of any one of claims 17 to 24, wherein at least one reagent for reverse transcriptase activity is one or more deoxyribonucleotide triphosphates.23 OFO-358193473

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