基于转录本捕获的宿主样本细菌特异性 RNA 富集方法

Eleanor I. Lamont; Richard M. Jones; Jessica Assadi; Shuyi Ma; David R. Sherman

doi:10.21769/BioProtoc.5712

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Peer-reviewed

Enriching Bacteria-Specific RNA From Host Samples Before NGS With Transcript-Capture

基于转录本捕获的宿主样本细菌特异性 RNA 富集方法

EL Eleanor I. Lamont ^§

RJ Richard M. Jones

JA Jessica Assadi

SM Shuyi Ma

DS David R. Sherman email

(§Technical contact: elamont@uw.edu) 发布: 2026年06月20日第16卷第12期 DOI: 10.21769/BioProtoc.5712 浏览次数: 331

评审: Alba BlesaJiadong ZhaoKyle Stewart Skalenko

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Abstract

Pathogen gene expression from host samples is often challenging to study due to low signal and high host RNA background. PCR probes have been recently used to hybridize and extract bacterial sequences from next-generation sequencing (NGS) libraries generated from in vitro and animal models of infection; however, these strategies require purchasing commercially synthesized probes that often do not capture the entire transcriptome. Transcript-capture sequencing is a novel capture approach for extracting RNA of a target bacterial species from samples in which there is substantial contamination by the host or other microbes. Biotinylated 150-base-pair DNA probes are generated in-house from bacterial DNA spanning the entire bacterial genome. Probes are hybridized to the cDNA of NGS sequencing libraries prepared from host samples to capture and enrich for bacterial-specific RNA reads before sequencing. This method results in a >200-fold increase in bacterial RNA reads from infected host samples (including in vitro, animal, and human samples) and generates complete bacterial transcriptomes with high gene coverage (>80%). Use of this protocol on infected host samples reveals a snapshot of bacterial activity during disease that may improve understanding of the physiological state of pathogens within their hosts.

Key features

• Generates single-stranded biotinylated DNA probes in-house from bacterial gDNA.

• Uses DNA probes in a hybridization capture approach to enrich for cDNA of a target species >200-fold in standard next-generation RNA sequencing libraries.

• Allows generation of complete bacterial transcriptomes (>80% gene coverage) from samples in which transcriptomic signal-to-noise is limiting, such as from animal models and clinical samples.

Keywords: DNA probes (DNA 探针)

Bacterial transcriptomics (细菌转录组学)

NGS (NGS)

Infected host samples (感染宿主样本)

Dot blot (斑点杂交)

Graphical overview

Background

Measuring messenger RNA (mRNA) from living bacteria is a promising method to understand the physiological state of pathogens within host environments. However, bacterial activity in vivo is not well described as it is technically difficult to process the minimal amount of bacterial mRNA present in most infected host material [1–4]. For example, previous work using dual-RNA next-generation sequencing (NGS) on clinical tuberculosis sputum samples has mapped only 0.85% (<300,000) bacterial reads to the infecting pathogen Mycobacterium tuberculosis (Mtb) due to an abundance of host RNA [5]. Several methods have previously been used to specifically enrich for bacterial RNA out of mixed samples for NGS applications, including using commercially synthesized biotinylated oligonucleotides that hybridize to matching sequences in an NGS sequencing library [6–10]. This strategy has limitations: custom probe design and synthesis is expensive, pre-designed probe libraries often fail to capture all genes present, and these methods have not been well-validated in clinical samples.

Here, we describe the transcript-capture sequencing protocol to enrich for bacterial RNA out of mixed samples, including animal models and clinical samples. Like the recently described DNA-capture technique genome capture sequencing (GenCap-Seq) [11,12], we produce biotinylated probes generated in-house from bacterial gDNA in a cost-effective and unbiased approach to sequence complete bacterial transcriptomes from infected host material. Using this protocol, we have generated complete bacterial transcriptomes (>1 million bacterial reads and >60% of bacterial genes having ≥10 reads aligning) from samples with as few as 1 × 10⁴ bacterial cells, with partial transcriptomes recovered from samples with as few as 1 × 10² cells [13]. We used probes generated from Mtb DNA for validation of transcript-capture; however, probe generation is customizable and can be used to determine the transcriptomes of other target species, with the potential for wide applications across different sample types and microbial species. For example, this protocol could be adapted to generate DNA probes of any microbial species or strain (including bacteria, viruses, parasites, or fungi) to enrich specific RNA from complex mixed samples such as infected host tissue, microbiome samples, or environmental sources like soil and seawater.

Materials and reagents

Biological materials

1. Bacterial DNA (see General notes 1 and 2)

2. NGS library for RNA sequencing prepared from bacterial-infected host sample (see General notes 3–5)

Reagents

1. DEPC H₂O (Sigma-Aldrich, catalog number: 693520)

2. 1 M Tris-HCl pH 8 (Fisher Scientific, catalog number: 15-568-025)

3. 5 M sodium chloride (NaCl) (Invitrogen, catalog number: AM9759)

4. 0.5 M EDTA (Invitrogen, catalog number: AM9261)

5. Saline sodium citrate (SSC) buffer, 20× concentrated (Sigma-Aldrich, catalog number: SRE0068)

6. Formamide (Thermo Scientific, catalog number: 17899)

7. Tween-20 (Sigma-Aldrich, catalog number: P1379)

8. Ampure XP beads (Beckman Coulter, catalog number: A63882)

9. Shrimp alkaline phosphatase (rSAP) and rCutSmart buffer (NEB, catalog number: M0371S)

10. Terminal transferase and cobalt (II) chloride (CoCl₂) (NEB, catalog number: M0315S)

11. Biotin-11-ddATP (Revvity, catalog number: NEL548001EA)

12. Qubit ssDNA Assay kit (ThermoFisher, catalog number: Q10212)

13. Qubit dsDNA HS Assay kit (ThermoFisher, catalog number: Q32851)

14. Monarch PCR & DNA Cleanup kit (NEB, catalog number: T1030)

15. Dynabeads MyOne Streptavidin C1 (Invitrogen, catalog number: 65002)

16. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A5611)

17. Streptavidin-AP conjugate (Roche, catalog number: 11089161001)

18. BCIP/NBT liquid substrate system (Sigma, catalog number: B1911-100ML)

19. xGen Human Cot DNA (IDT, catalog number: 1080768)

20. xGen Universal blockers (IDT, catalog number: 1075475)

21. xGen Hybridization and Wash v2 kit (IDT, catalog number: 1080584)

22. Dynabeads M-270 Streptavidin (Fisher, catalog number: 65-305)

23. 200 proof ethanol (EtOH) (Sigma-Aldrich, catalog number: E7023)

24. Nuclease-free H₂O (Invitrogen, catalog number: 10977015)

25. 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog number: KK2602)

26. P5 primer 5′ AATGATACGGCGACCACCGA 3′ ordered from IDT at 25 nmol concentration and purified with standard desalting

27. P7 primer 5′ CAAGCAGAAGACGGCATACGA 3′ ordered from IDT at 25 nmol concentration and purified with standard desalting

Solutions

1. 10 μM Biotin-11-ddATP (see Recipes)

2. 2× binding and wash buffer (see Recipes)

3. 1× binding and wash buffer (see Recipes)

4. 0.1× SSC (see Recipes)

5. Formamide solution (see Recipes)

6. 10× Tris-buffered saline (TBS) (see Recipes)

7. 1× Tris-buffered saline with Tween-20 (TBST) (see Recipes)

8. Blocking buffer (see Recipes)

9. 1:2,000 Streptavidin-AP conjugate (see Recipes)

10. 80% EtOH (see Recipes)

11. 95% EtOH (see Recipes)

12. 10 mM Tris-HCl, pH 8.5 (see Recipes)

Recipes

1. 10 μM Biotin-11-ddATP

Reagent	Final concentration	Quantity or volume
Biotin-11-ddATP	10 μM	1 μL
DEPC H₂O		99 μL

Store at 4 °C for up to 1 year.

2. 2× binding and wash buffer

Reagent	Final concentration	Quantity or volume
1 M Tris-HCl	10 mM	500 μL
5 M NaCl	300 mM	3 mL
0.5 M EDTA	1 mM	100 μL
DEPC H₂O		46.4 mL

Sterilize by filtering through a 0.22 μm filter. Store at room temperature.

3. 1× binding and wash buffer

Reagent	Final concentration	Quantity or volume
2× binding and wash buffer	1×	10 mL
DEPC H₂O		10 mL

Store at room temperature.

4. 0.1× SSC

Reagent	Final concentration	Quantity or volume
20× SSC	0.1×	250 μL
DEPC H₂O		49.75 mL

Sterilize by filtering through a 0.22 μm filter. Store at room temperature.

5. Formamide solution

Reagent	Final concentration	Quantity or volume
Formamide	95%	950 μL
0.5 M EDTA	10 mM	20 μL
DEPC H₂O		30 μL

Aliquot 100 μL into 1.5 mL tubes. Store at -20 °C. Avoid freeze-thawing.

6. 10× TBS

Reagent	Final concentration	Quantity or volume
1 M Tris-HCl pH 8	200 mM	100 mL
5 M NaCl	1.5 M	150 mL
DEPC H₂O		250 mL

Filter through a 0.22 μm filter to sterilize. Store at room temperature.

7. 1× TBST

Reagent	Final concentration	Quantity or volume
10× TBS	1×	50 mL
Tween 20	0.1%	500 μL
DEPC H₂O		450 mL

Filter through a 0.22 μm filter to sterilize. Store at room temperature.

8. Blocking buffer

Reagent	Final concentration	Quantity or volume
BSA	2%	10 g
1× TBST		500 mL

Filter through a 0.22 μm filter to sterilize. Store at room temperature.

9. 1:2,000 Streptavidin-AP conjugate

Reagent	Final concentration	Quantity or volume
Streptavidin-AP conjugate	1:2,000	7.5 μL
Blocking buffer		15 mL

Prepare immediately before use. Do not store.

10. 80% EtOH

Reagent	Final concentration	Quantity or volume
200 proof EtOH	80%	40 mL
DEPC H₂O		10 mL

Filter through a 0.22 μm filter to sterilize. Store at room temperature and use within 1 week.

11. 95% EtOH

Reagent	Final concentration	Quantity or volume
200 proof EtOH	95%	47.5 mL
DEPC H₂O		2.5 mL

Filter through a 0.22 μm filter to sterilize. Store at room temperature and use within 1 week.

12. 10 mM Tris-HCl, pH 8.5

Reagent	Final concentration	Quantity or volume
1 M Tris-HCl	10 mM	100 μL
DEPC H₂O		9.9 mL

Check pH with a pH reader and adjust to pH 8.5. Filter through a 0.22 μm filter to sterilize. Store at room temperature.

Laboratory supplies

1. Covaris microTUBE AFA fiber pre-slit snap-cap 6 × 16 mm (Fisher Scientific, catalog number: NC9871616)

2. FlashGel system (Lonza, catalog number: 57067)

3. 1.5 mL DNA LoBind tubes (Eppendorf, catalog number: 0030108051)

4. 5 mL DNA LoBind tubes (Eppendorf, catalog number: 0030108310)

5. 96-well LoBind PCR plates (Eppendorf, catalog number: 0030129512)

6. 0.2 mL PCR 8-strip tubes (VWR, catalog number: 490003-692)

7. Magnetic Stand-96 (Invitrogen, catalog number: AM10027)

8. DynaMag-2 magnet (Invitrogen, catalog number: 12321D)

9. 0.22 μm Stericup vacuum filtration system (Fisher Scientific, catalog number: S2GPU05RE)

10. 0.2 μm nitrocellulose membrane (Thermo Scientific, catalog number: 88024)

11. Microseal B PCR plate sealing film (Bio-Rad, catalog number: 17010701)

12. 20 μL BIOTIX XTIP Rainin LTS compatible filter low-retention sterile pipette tips (Fisher Scientific, catalog number: 12-111-400)

13. 200 μL BIOTIX XTIP Rainin LTS compatible filter low-retention sterile pipette tips (Fisher Scientific, catalog number: 12-111-362)

14. 1,000 μL BIOTIX XTIP Rainin LTS compatible filter low-retention sterile pipette tips (Fisher Scientific, catalog number: 12-111-364)

Equipment

1. Pipet-Lite LTS Pipette L-2×LS+ manual single-channel pipette, 0.1–2 μL (Mettler Toledo, catalog number: 17014393)

2. Pipet-Lite LTS Pipette L-20×LS+ manual single-channel pipette, 2–20 μL (Mettler Toledo, catalog number: 17014392)

3. Pipet-Lite LTS Pipette L-20×LS+ manual single-channel pipette, 20–200 μL (Mettler Toledo, catalog number: 17014391)

4. Pipet-Lite LTS Pipette L-20×LS+ manual single-channel pipette, 100–1,000 μL (Mettler Toledo, catalog number: 17014382)

5. Pipet-Lite XLS+ manual 12-channel pipette 2–20 μL (Mettler Toledo, catalog number: 17013808)

6. Pipet-Lite XLS+ manual 12-channel pipette 20–200 μL (Mettler Toledo, catalog number: 17013805)

7. M220 Focused-ultrasonicator (Covaris, catalog number: 500295)

8. Microcentrifuge (Eppendorf, catalog number: 13-864-454)

9. Dry block heater (VWR, catalog number: 75838-318)

10. Qubit 4 Fluorometer (ThermoFisher, catalog number: Q33238)

11. Two 96-well thermal cyclers (Bio-Rad, catalog number: 1861096)

12. Vortex mixer (VWR, catalog number: 97043-562)

13. Benchtop laboratory pH/mV meter (Fisher Scientific, catalog number: 13636AB315A)

14. UV crosslinker (Fisher Scientific, catalog number: 13-245-221)

15. Low-speed orbital shaker (Corning, catalog number: CLS6782FP)

16. SpeedVac integrated vacuum concentrator system (Thermo Scientific, catalog number: 1156U31)

17. Freezer (-20 °C)

18. Refrigerator (4 °C)

Procedure

English

中文翻译

文章信息

稿件历史记录

提交日期: Feb 27, 2026

接收日期: Apr 27, 2026

在线发布日期: May 19, 2026

出版日期: Jun 20, 2026

版权信息

如何引用

Lamont, E. I., Jones, R. M., Assadi, J., Ma, S. and Sherman, D. R. (2026). Enriching Bacteria-Specific RNA From Host Samples Before NGS With Transcript-Capture. Bio-protocol 16(12): e5712. DOI: 10.21769/BioProtoc.5712.