AQRNA-seq for Quantifying Small RNAs

Summary

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

Abstract

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

Introduction

Next-generation sequencing (NGS), also known as massively parallel sequencing, is a DNA sequencing technology that involves DNA fragmentation, ligation of adaptor oligonucleotides, polymerase chain reaction (PCR)-based amplification, sequencing of the DNA, and reassembly of the fragment sequences into a genome. The adaptation of NGS to sequence RNA (RNA-seq) is a powerful approach to identify and quantify RNA transcripts and their variants¹. Innovative developments in RNA library preparation workflows and bioinformatic analysis pipelines, coupled with advancements in laboratory instrumentation, have expanded the repertoire of RNA-seq applications, progressing beyond exome sequencing into advanced functional omics like non-coding RNA profiling², single cell analysis³, spatial transcriptomics^4,5, alternative splicing analysis⁶, among others. These advanced RNA-seq methods reveal complex RNA functions through quantitative analysis of the transcriptome in normal and diseased cells and tissue.

Despite these advances in RNA-seq, several key technical features limit the quantitative power of the method. While most RNA-seq methods allow precise and accurate quantification of changes in the levels of RNAs between experimental variables (i.e., biological samples and/or physiological states), they cannot provide quantitative comparisons of the levels of RNA molecules within a sample. For example, most RNA-seq methods cannot accurately quantify the relative number of copies of individual tRNA isoacceptor molecules in a cellular pool of expressed tRNAs. As highlighted in the companion publication⁷, this limitation to RNA-seq arises from several features of RNA structure and the biochemistry of library preparation. For example, the activity of the ligation enzymes used to attach the 3'- and 5'-end sequencing linkers to RNA molecules is strongly influenced by the identity of the terminal nucleotides of the RNA and the sequencing linkers. This leads to large variations in efficiencies of linker ligations and profound artifactual increases in sequencing reads^8,9,10.

A second set of limitations arise from the inherent structural properties of RNA molecules. Specifically, RNA secondary structure formation and dynamic changes in the dozens of post-transcriptional RNA modifications of the epitranscriptome can cause polymerase fall-off or mutation during reverse transcription. These errors result in incomplete or truncated cDNA synthesis or altered RNA sequence. While both of these phenomena can be exploited to map secondary structures or some modifications, they degrade the quantitative accuracy of RNA-seq if subsequent library preparation steps fail to capture truncated cDNAs or if data processing throws out mutated sequences not matching a reference dataset^11,12. Furthermore, the immense chemical, length, and structural diversity of RNA transcripts, as well as the lack of tools to uniformly fragment long RNAs, diminishes the applicability of most RNA-seq methods to all RNA species¹³.

The AQRNA-seq (absolute quantification RNA sequencing) method has been developed to remove several of these technical and biological constraints that limit quantitative accuracy⁷. By minimizing sequence-dependent biases in capture, ligation, and amplification during RNA sequencing library preparation, AQRNA-seq achieves superior linearity compared to other methods, accurately quantifying 75% of a reference library of 963 miRNAs within 2-fold accuracy. This linear correlation of sequencing read count and RNA abundance is also observed in an analysis of a variable length pool of RNA oligonucleotide standards and in reference to orthogonal methods like northern blotting. Establishing linearity between sequencing read count and RNA abundance enables AQRNA-seq to achieve accurate, absolute quantification of all RNA species within a sample.

Here is a description of the protocol for AQRNA-seq library preparation workflow and the accompanying downstream data analytics pipeline. The method was applied to elucidate the dynamics of tRNA abundance during starvation-induced dormancy and subsequent resuscitation in the Mycobacterium bovis bacilli de Calmette et Guérin (BCG) model of tuberculosis. Results were presented for the exploratory visualization of the sequencing data, along with subsequent clustering and differential expression analyses that unveiled discernable patterns in tRNA abundance associated with various phenotypes.

Protocol

NOTE: Figure 1 provides a graphical illustration of the procedures involved in AQRNA-seq library preparation. Detailed information regarding the reagents, chemicals, and columns/kits used in the procedure can be found in the Table of Materials. It is recommended to perform a comprehensive evaluation of purity, integrity, and quantity of the input RNA samples using (i) 3% agarose gel electrophoresis, (ii) automated electrophoresis tools for sample quality control of biomolecu…

Representative Results

Mycobacterium bovis BCG (bacilli de Calmette et Guérin) strain 1173P2 undergoing exponential growth were subject to a time series (0, 4, 10, and 20 days) of nutrient starvation, followed by a 6-day resuscitation in nutrient-rich medium as previously presented in Hu et al.7. Small RNAs were isolated from bacterial culture, with three biological replicates, at each of the five designated time points. Illumina libraries were constructed using the above-described AQRNA-seq library prepar…

Discussion

The AQRNA-seq library preparation workflow is designed to maximize the capture of RNAs within a sample and minimize polymerase fall-off during reverse transcription⁷. Through a two-step linker ligation, novel DNA oligos (Linker 1 and Linker 2) are ligated in excess to fully complement the RNA within the sample. Excess linkers can be efficiently removed with RecJf, a 5' to 3' exonuclease specific to single-stranded DNAs, leaving the ligated products intact. In addition, AlkB treatment reduc…

Materials

2-ketoglutarate	Sigma-Aldrich	75890	Prepare a working solution (1 M) and store it at -20ºC
2100 Bioanalyzer Instrument	Agilent	G2938C
5'-deadenylase (50 U/μL)	New England Biolabs	M0331S (component #: M0331SVIAL)	Store at -20 °C
Adenosine 5'-Triphosphate (ATP)	New England Biolabs	M0437M (component #: N0437AVIAL)	NEB M0437M contains T4 RNA Ligase 1 (30 U/μL), T4 RNA Ligase Reaction Buffer (10X), PEG 8000 (1X), and ATP (100 mM); prepare a working solution (10 mM) and store it at -20ºC
AGAROSE GPG/LE	AmericanBio	AB00972-00500	Store at ambient temperature
Ammonium iron(II) sulfate hexahydrate	Sigma-Aldrich	F2262	Prepare a working solution (0.25 M) and store it at -20 °C
Bioanalyzer Small RNA Analysis	Agilent	5067-1548	The Small RNA Analysis is used for checking the quality of input RNAs and the efficiency of enzymatic reactions (e.g., Linker 1 ligation)
Bovine Serum Albumin (BSA; 10 mg/mL)	New England Biolabs	B9000	This product was discontinued on 12/15/2022 and is replaced with Recombinant Albumin, Molecular Biology Grade (NEB B9200).
Chloroform	Macron Fine Chemicals	4441-10
Demethylase	ArrayStar	AS-FS-004	Demethylase comes with the rtStar tRNA Pretreatment & First-Strand cDNA Synthesis Kit (AS-FS-004)
Deoxynucleotide (dNTP) Solution Mix	New England Biolabs	N0447L (component #: N0447LVIAL)	This dNTP Solution Mix contains equimolar concentrations of dATP, dCTP, dGTP and dTTP (10 mM each)
Digital Dual Heat Block	VWR Scientific Products	13259-052	Heating block is used with the QIAquick Gel Extraction Kit
DyeEx 2.0 Spin Kit	Qiagen	63204	Effective at removing short remnants (e.g., oligos less than 10 bp in length)
Electrophoresis Power Supply	Bio-Rad Labrotories	PowerPac 300
Eppendorf PCR Tubes (0.5 mL)	Eppendorf	0030124537
Eppendorf Safe-Lock Tubes (0.5 mL)	Eppendorf	022363611
Eppendorf Safe-Lock Tubes (1.5 mL)	Eppendorf	022363204
Eppendorf Safe-Lock Tubes (2 mL)	Eppendorf	022363352
Ethyl alcohol (Ethanol), Pure	Sigma-Aldrich	E7023	The pure ethanol is used with the Oligo Clean and Concentrator Kit from Zymo Research
Gel Imaging System	Alpha Innotech	FluorChem 8900
Gel Loading Dye, Purple (6X), no SDS	New England Biolabs	N0556S (component #: B7025SVIAL)	NEB N0556S contains Quick-Load Purple 50 bp DNA Ladder and Gel Loading Dye, Purple (6X), no SDS
GENESYS 180 UV-Vis Spectrophotometer	Thermo Fisher Scientific	840-309000	The spectrophotometer is used for measuring the oligo concentrations using the Beer's law
HEPES	Sigma-Aldrich	H4034	Prepare a working solution (1 M; pH = 8 with NaOH) and store it at -20 °C
Hydrochloric acid (HCl)	VWR Scientific Products	BDH3028	Prepare a working solution (5 M) and store it at ambient temperature
Isopropyl Alcohol (Isopropanol), Pure	Macron Fine Chemicals	3032-16	Isopropanol is used with the QIAquick Gel Extraction Kit
L-Ascorbic acid	Sigma-Aldrich	A5960	Prepare a working solution (0.5 M) and store it at -20ºC
Microcentrifuge	Eppendorf	5415D
NanoDrop 2000 Spectrophotometer	Thermo Fisher Scientific	ND-2000
NEBuffer 2 (10X)	New England Biolabs	M0264L (component #: B7002SVIAL)	NEB M0264L contains RecJf (30 U/μL) and NEBuffer 2 (10X); store at -20 °C
Nuclease-Free Water (not DEPC-Treated)	Thermo Fisher Scientific	AM9938
Oligo Clean & Concentrator Kit	Zymo Research	D4061	Store at ambient temperature
PEG 8000 (50% solution)	New England Biolabs	M0437M (component #: B1004SVIAL)	NEB M0437M contains T4 RNA Ligase 1 (30 U/μL), T4 RNA Ligase Reaction Buffer (10X), PEG 8000 (1X), and ATP (100 mM); prepare a working solution (10 mM) and store it at -20ºC
Peltier Thermal Cycler	MJ Research	PTC-200
Phenol:choloroform:isoamyl alcohol 25:24:1 pH = 5.2	Thermo Fisher Scientific	J62336
PrimeScript Buffer (5X)	TaKaRa	2680A
PrimeScript Reverse Transcriptase	TaKaRa	2680A
QIAquick Gel Extraction Kit	Qiagen	28704	This kit requires a heating block and isopropanol to work with
Quick-Load Purple 100 bp DNA Ladder	New England Biolabs	N0551S (component #: N0551SVIAL)
Quick-Load Purple 50 bp DNA Ladder	New England Biolabs	N0556S (component #: N0556SVIAL)	NEB N0556S contains Quick-Load Purple 50 bp DNA Ladder and Gel Loading Dye, Purple (6X), no SDS
RecJf (30 U/μL)	New England Biolabs	M0264L (component #: M0264LVIAL)	NEB M0264L contains RecJf (30 U/μL) and NEBuffer 2 (10X); store at -20 °C
RNase Inhibitor (murine; 40 U/μL)	New England Biolabs	M0314L (component #: M0314LVIAL)	Store at -20 °C
SeqAMP DNA Polymerase	TaKaRa	638509	TaKaRa 638509 contains SeqAMP DNA Polymerase and SeqAMP PCR Buffer (2X)
SeqAMP PCR Buffer (2X)	TaKaRa	638509	TaKaRa 638509 contains SeqAMP DNA Polymerase and SeqAMP PCR Buffer (2X)
Shrimp Alkaline Phosphatase (1 U/μL)	New England Biolabs	M0371L (component #: M0371LVIAL)
Sodium hydroxide (NaOH)	Sigma-Aldrich	S5881	Prepare a working solution (5 M) and store it at ambient temperature
T4 DNA Ligase (400 U/μL)	New England Biolabs	M0202L (component #: M0202LVIAL)	NEB M0202L contains T4 DNA Ligase (400 U/μL) and T4 DNA Ligase Reaction Buffer (10X)
T4 DNA Ligase Reaction Buffer (10X)	New England Biolabs	M0202L (component #: B0202SVIAL)	NEB M0202L contains T4 DNA Ligase (400 U/μL) and T4 DNA Ligase Reaction Buffer (10X)
T4 RNA Ligase 1 (30 U/μL)	New England Biolabs	M0437M (component #: M0437MVIAL)	NEB M0437M contains T4 RNA Ligase 1 (30 U/μL), T4 RNA Ligase Reaction Buffer (10X), PEG 8000 (1X), and ATP (100 mM)
T4 RNA Ligase Reaction Buffer (10X)	New England Biolabs	M0437M (component #: B0216SVIAL)	NEB M0437M contains T4 RNA Ligase 1 (30 U/μL), T4 RNA Ligase Reaction Buffer (10X), PEG 8000 (1X), and ATP (100 mM)