Chez Charles/ tags/ pages tagged template_switching

(work in progress. For alternative cap enrichement methods see the cap tag page)

Methods papers.

  • 1996: ”Synthesis of high-quality cDNA from nanograms of total or polyA+ RNA with the CapFinder PCR cDNA library construction kit.” Zhu, Y., A. Chenchik and P.D. Siebert. CLONTECHniques 1 1:12-13. Could not find the PDF.

  • 1998: “Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR” Chenchick and coll., 1998. Oligo-dT-primed total RNA is template-switched with rGrGrG DNA/RNA hybrids. SMART means “Switch Mechanism At the 5′ end of RNA Templates”. Is that the primary paper for SMART ?

  • In the "CapSelect" method, Schmidt and Mueller, 1999 stimulate template switching with manganese (see below), tail the first-strand cDNAs with dA, and add 5′ linkers with T4 DNA ligase and duplex adapters ending with a (T)TTTGGG overhang.

  • Noticing that the length of the dC tail on the first-strand cDNA is varialbe, Shi and Kaminskyj (2000) prepared collections of TSOs with variable rG tail length, using TdT.

  • In SMART (switching mechanism at the 5′ end of the RNA transcript), Zhu, Machleder, Chenchik, Li and Siebert (2001) , first-strand cDNAs are prepared with rGrGrG TSOs containing a SfiIB site and oligo-dT RT primers containing a SfiIA site. Second-strand synthesis with low-cycle PCR, followed with standard cloning methods.

  • The "terminal continuation" method (Ginsberg et al., 2002, Che et al., 2004) is essentially a template switching with DNA oligonucleotides ending in CCC or GGG. AAA and TTT were also tested. An extensive protocol was published in Ginsberg, 2005.

  • In the single-cell tagged reverse transcription (STRT) method, Islam et al, (2011) use template switching and unique molecular identifiers to sequence 5′ ends. The method is oligo-dT-primed.

  • Mohr et al (2013) have shown (Fig 6) that retroviral RTs can extend a linker with the sequence of a small RNA via a template switching reaction. (That is: a sRNA can play the same role as a TS oligonucleotide.)

  • in Capture and Amplification by Tailing and Switching (CATS, Turchinovich et al (2014)), short and long RNAs are A-tailed, oligo-dT-primed, and template swiched. A PNK treatement is needed on circulating RNAs, to remove phosphates or cyclophosphates that would prevent the A-tailing.

  • in nanoPARE (Schon, Kellner and coll.), a template-switching oligonucleotide (RNA-RNA-LNA, without UMIs) is used to add a linker on 5′ ends. After tagmentation, two libraries are amplified: one for 5′ ends and one for RNA-seq. ~15% of the 5′ end alignments have extra Gs, but the genomic distribution is bimodal. Peaks with significant amounts of "extra G" nucleotides are marked as TSS.

  • ?Policastro and coll, 2020 and others before them add the template-switching oligonucleotide after the reverse-transription has been incubated for some time.

Effect of chemical composition of the TS oligonucleotide

Originally, the TSOs were all-RNA. Since this is expensive to synthesise, TSOs where only the last 3 bases are RNA became popular. LNA was also tested as a replacement for RNA.

  • Chenchick and coll., 1998 reported (rG)n >> rG > dGdGdG >> rUrUrU.

  • Picelli et al (2013) reported a higher performance for RRL compared to RRR, when preparing Smart-seq2 libraries.

  • Harbers et al (2013) used the nanoCAGE protocol to compare TSOs ending in RRR, DDD, DDL, DLL or LLL, and reported that only the RRR TSOs had good efficiency (less PCR cycles needed to amplify the cDNAs) and had the lowest amount of strand invastion artefacts.

  • Arguel et al (2017) reported similar performance for RRR and RRL, using a 5′-focused method similar to nanoCAGE or STRT.

  • 3′ phosphate or biotin blocking groups abolish template-switching (Turchinovich et al (2014) and others). However, Pinto & Lindblad (2010) report the use of a 3′ C3 spacer (on all-DNA TSOs) and Dai and coll. 2020. report successful use of a phosphorylated TSO.

  • 5′ iso-dC and iso-dG prevents reverse-transcriptase to reach the end of the TSO, and therefore blocks concatenation (Kapteyn et al., 2010). This was also used in FFPEcap-seq (Vahrenkamp and coll., 2019).

Effect of TSO concentration

  • For the STRT method, Zajac et al (2013) concluded that 1 μM of TSO gave the highest yield.

Effect of magnesium, manganese and dNTP concentrations

  • Schmidt and Mueller, 1999 showed that increasing magnesium concentration (to 6 mM) or adding manganese at the end of the reaction (1 or 2 mM) increased the frequency of dC addition (moderately for Mg2+ and strongly for Mn2+). Enzyme: SSII; dNTP concentration: 1 mM each. Pinto & Lindblad (2010) also used manganese.

  • Lee and coll. (2017) increased the efficiency of template switching non-capped molecules by increasing dNTPs to 2 mM and Mg2+ to 9 mM.

  • Vahrenkamp and coll. (2019) reported that addition of 1 mM manganese increases the formation of TSO concatenates and the fraction of reads aliging to ribosomal sequences.

Related works

  • The AMV RT was reported by Ouhammouch and Brody (1992) to template-switch from a mRNA to a plasmid.
RSS Atom
Fidelity of in vitro DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase.

Peliska JA, Benkovic SJ.

Biochemistry. 1994 Apr 5;33(13):3890-5 doi:10.1021/bi00179a014

Fidelity of in vitro DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase.

When the extra base added by the RTase in a template-switching reaction is determined by sequencing, the result does not reflect previous results obtained by primer extension assay (where A >> C on non-capped blunt DNA/RNA ends). This may be because of the differential efficiency of the RTase to extend a template over various single-base mismatches. In this assay, T to C and G to C were more frequent than T to A and G to A. Nevertheless, the experiments support previous evidence that addition is mostly limited to a single nucleotide. RT reaction: 50 mM Tris-HCl, pH 8.0; 75 mM KCl;, 0.1 mM EDTA; 1 mM DTT; 0.1% Triton X-100; 100 µM each dNTP; 7 mM MgCl2; 200 nM 24-base DNA-40-base RNA primer-template; 700 nM 41-base RNA template 2; 700 nM 42-base RNA template 3; and 100 nM HIV-1 RT in a final volume of 10 µL. When reaction products were to be sequenced, mixtures were incubated at 37 °C for 2 h.

Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR.

Alex Chenchick, York Y. Shu, Luda Diatchenko, Roger Li, Jason Hill and Paul D. Siebert. (Gene Cloning and Analysis Group, CLONETECH Laboratories, Pao Alto, CA, USA).

In: Gene Cloning and Analysis by RT-PCR. Edited by Paul Siebert and James Larrick. 1998

Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR.

Reaction mixture: 1 µM RTP; 1 µM TSO; 50–1000 ng total RNA; 2 mM DTT, 1 mM dNTP, 200 U SSII in 10 µL.

DNA/RNA ends tested: HO-G, Cap-G, HO-A, Cap-A, HO-C, Cap-C, HO-T

TSOs tested: rG, rGrG, rGrGrG, rGrGrGrGrG, rUrUrU, GGG, rGrGrG in all-r oligo.

With the wild-type MMLVm the HO-G DNA/RNA duplex is tailed with 1~5 extra nucleotides (Fig 2). Using radiolabelled nucleotides suggests that they are mostly Cs. "Not shown" experiments suggest that the presence of a cap "does not significantly influence the preference of addition of these non-templated nucleotides". The consensus tail is AACCC. SSII (RNAseH-) has a lower efficiency for adding nucleotides, compared with wild-type MMLV.

Template-switching is more efficient with at least 2 rG. dG is notably less efficient and rU has no visible efficiency (Figure 2).

In 2 % of the cDNAs, RT was primed by the TSO.

5' RACE by tailing a general template-switching oligonucleotide.

Shi X, Kaminskyj SG.

Biotechniques. 2000 Dec;29(6):1192-5 doi:10.2144/00296bm07

5' RACE by tailing a general template-switching oligonucleotide.

Generate a collection of TSOs by TdT tailing DNA oligos with GTP. As the lengh of the G tail is variable, it is thought to be more suited to the template switching on cDNAs where the C tail is also variable in length.

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Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction.

Biotechniques. 2001 Apr;30(4):892-7.

Zhu YY, Machleder EM, Chenchik A, Li R, Siebert PD.

Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction.

SMART: “switching mechanism at the 5′ end of the RNA transcript”. Template-swiching with a rGrGrG oligo containing SfiIB, reverse transcription with an oligo-dT primer containing a SfiIA site. Second-strand cDNA synthesis with low-cycle PCR. Cloning with standard methods.

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NanoPARE: parallel analysis of RNA 5' ends from low-input RNA.

Genome Res. 2018 Dec;28(12):1931-1942. doi:10.1101/gr.239202.118

Schon MA, Kellner MJ, Plotnikova A, Hofmann F, Nodine MD.

NanoPARE: parallel analysis of RNA 5' ends from low-input RNA.

5′ ends tags and RNA-seq tags separately amplified in two libraries. Non-TSS peaks (identify by low-extraG content) contain miRNA cleavage sites.

STRT-seq-2i: dual-index 5' single cell and nucleus RNA-seq on an addressable microwell array.

Sci Rep. 2017 Nov 27;7(1):16327. doi:10.1038/s41598-017-16546-4

Hochgerner H, Lönnerberg P, Hodge R, Mikes J, Heskol A, Hubschle H, Lin P, Picelli S, La Manno G, Ratz M, Dunne J, Husain S, Lein E, Srinivasan M, Zeisel A, Linnarsson S.

STRT-seq-2i: dual-index 5' single cell and nucleus RNA-seq on an addressable microwell array.

STRT for the icell8 platform. SSIII better than SSIII with RNA TSO. Optimum: ~2 μM.

Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples.

BMC Genomics. 2010 Jul 2;11:413. doi:10.1186/1471-2164-11-413

Kapteyn J, He R, McDowell ET, Gang DR.

Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples.

TSO starting with iCiGiC (iso-dC / iso-dG) does not form concatenates because the RTase is inhibited by these nucleotides, therefore it does not reach the end and no extra template switching occurs.

Capture and Amplification by Tailing and Switching (CATS). An ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA.

RNA Biol. 2014;11(7):817-28. doi:10.4161/rna.29304

Turchinovich A, Surowy H, Serva A, Zapatka M, Lichter P, Burwinkel B.

Capture and Amplification by Tailing and Switching (CATS). An ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA.

Pre-treatment by T4 PNK was needed when using circulating plasma RNA, probably because of phosphate or cyclo-phosphates at the 3′ end (caused by RNA cleavage). RT primer concentration scaled down according to the RNA amoounts (0.1 µM for 1 ng and 0.1 nM for 5 pg). TSO concentration: 1 µM.

Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium.

Lee YH, Hsueh YW, Peng YH, Chang KC, Tsai KJ, Sun HS, Su IJ, Chiang PM.

BMC Biol. 2017 Mar 21;15(1):22. doi:10.1186/s12915-017-0359-5

Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium.

Poly-A tailing followed by template switching. Increased dNTPs to 2 mM and Mg2+ to 9 mM to favour terminal addition of nucleotides. TSO and RT primers are at 1 μM final.

A cost effective 5' selective single cell transcriptome profiling approach with improved UMI design.

Nucleic Acids Res. 2016 Dec 9. pii: gkw1242. doi:10.1093/nar/gkw1242

Arguel MJ, LeBrigand K, Paquet A, Ruiz García S, Zaragosi LE, Barbry P, Waldmann R.

A cost effective 5' selective single cell transcriptome profiling approach with improved UMI design.

Backload of barcodes between RT and PCR. Changing the terminal base of the template-switching oligonucleotide from RNA to LNA did not increase performance.

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

Fu GK, Wilhelmy J, Stern D, Fan HC, Fodor SP.

Anal Chem. 2014 Mar 18;86(6):2867-70. doi:10.1021/ac500459p

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

only 14 to 23 % of the cDNAs were template-switched.

Four methods of preparing mRNA 5' end libraries using the illumina sequencing platform.

Machida RJ, Lin YY.

PLoS One. 2014 Jul 8;9(7):e101812. doi: 10.1371/journal.pone.0101812

Four methods of preparing mRNA 5' end libraries using the illumina sequencing platform.

Capture of 5′ ends via biotinylated forward PCR primers. CapSMART: ligation of modified oligonucleotides that block RT on 5′-phoshphorylated RNAs (after kinase treatment). The signal is not much different from standard SMART.

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

Mohr S, Ghanem E, Smith W, Sheeter D, Qin Y, King O, Polioudakis D, Iyer VR, Hunicke-Smith S, Swamy S, Kuersten S, Lambowitz AM.

RNA. 2013 Jul;19(7):958-70. doi: 10.1261/rna.039743.113

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

Also reports the addition of a non-templated A.

High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression.

Batut P, Dobin A, Plessy C, Carninci P, Gingeras TR.

Genome Res. 2013 Jan;23(1):169-80. doi: 10.1101/gr.139618.112. Epub 2012 Aug 30.

High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression.

Combines three methods for cap specificity.

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Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells.

Ramsköld D, Luo S, Wang YC, Li R, Deng Q, Faridani OR, Daniels GA, Khrebtukova I, Loring JF, Laurent LC, Schroth GP, Sandberg R.

Nat Biotechnol. 2012 Jul 22. doi: 10.1038/nbt.2282.

Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells.

If it really uses the SMARTer kit, then their methods section is wrong and they fail to disclose the real sequence of their template-switching oligonucleotide.

Integrating whole transcriptome assays on a lab-on-a-chip for single cell gene profiling.

Bontoux N, Dauphinot L, Vitalis T, Studer V, Chen Y, Rossier J, Potier MC.

Lab Chip. 2008 Mar;8(3):443-50

Integrating whole transcriptome assays on a lab-on-a-chip for single cell gene profiling.

For simple RT-PCR, cDNAs were pre-amplified 40× on chip. cDNAs must be dilluted at least 500× before amplification, otherwise TS-PCR is inhibited by leftovers of the RT.

Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases.

Nucleic Acids Res. 1988 Oct 25;16(20):9677-86

Clark JM

Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases.

Terminal desoxynucleotidyl transferase activity of DNA polymerases, including AMV reverse transcriptase. Preference for adding As. ‘We cannot exclude the formal possibility that some of these latter events, particularly the addition of dCMP by AMV reverse transcriptase, involve the use of coding information made available as a result of a transient misalignment of the primer/template substrate.’

Reverse transcriptases can clamp together nucleic acids strands with two complementary bases at their 3'-termini for initiating DNA synthesis.

Oz-Gleenberg I, Herschhorn A, Hizi A.

Nucleic Acids Res. 2011 Feb;39(3):1042-53. doi: 10.1093/nar/gkq786

Reverse transcriptases can clamp together nucleic acids strands with two complementary bases at their 3'-termini for initiating DNA synthesis.

AT-rich cDNA tails are not triggering template-switching well. CC are the strongest. Mn₂⁺ potentiates template switching for MMLV. dGTP stabilises the clamp between the enzyme and the nucleic acids.

CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.

Nucleic Acids Res. 1999 Nov 1;27(21):e31.

Schmidt WM, Mueller MW.

CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.

Extra cytosine are more frequently added in presence of the 5′ cap. Increasing Mg2+ to 6 mM increases the frequency of addition of more than 1 C, but only moderately. Instead, when adding BSA in the reaction, supplementing it with 1 or 2 mM Mn2+ after 1h, 3-4 extra dC residues are added to most of the first strand cDNAs. Standard reaction: 0.75 μM oligo dT (or 0.25 μM gene-specific RT primer); 50 mM Tris-HCl (pH 8.3); 75 mM KCl; 3 mM MgCl2; 5 mM DTT; dNTPs 1mM each; 0.1 mg BSA; 20 U RNAse inhibitor (Roche), 200 U SuperScript II; 1h at 42 °C.

PCR-suppression effect: kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples.

Dai ZM, Zhu XJ, Chen Q, Yang WJ.

J Biotechnol. 2007 Feb 20;128(3):435-43. doi:10.1016/j.jbiotec.2006.10.018

PCR-suppression effect: kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples.

PCR amplification of a GeneRuler 1kb DNA Ladder (Fermentas) on which adatpers were ligated. Investigates “parameters which affect ITR self-annealing: (i) the length of PCR products [...] (ii) Primer concentration [...] (iii) The ratio of ITR length to primer length [and] (iv) [annealing temperature]”. Uses a 3′ phosphate to prevent the template switching oligonucleotide to prime a first strand cDNA.

“The 10 µl final [RT] reaction mixture contained 50 mM Tris–Cl (pH 8.3 at 25°C), 75 mM KCl,6 mM MgCl2, 2 mM MnCl2, 0.2 mg/ml BSA, 10 mM DTT, 1 mM dNTPs, 1µM TS-oligo, 1µM oligo(dT) adaptor (, 10 U RNase Inhibitor, and 150 U SuperScript II [and] was incubated at 42 °C for 1 h, followed by 45 °C for 30 min, and 50 °C for 10 min.”

“Using [...] the shortest adaptor, whose length is equal to [the PCR primer], short fragments corresponding to 0.25 kb were efficiently amplified, while longer fragments (>3.5 kb) could not be distinguished from the background. Using [...] the longest adaptor, whose length is more than twice [the PCR primer], fragments shorter than 1.5 kb were suppressed, while fragments up to 10 kb were efficiently amplified.” [My comment: this analysis does not take into account the overall reduction of PCR efficiency with increasing and the fact that mass of shortest fragments were also lower in the GeneRuler ladder. This also contributes to the disappearance of these bands on the electrophoresis pictures. This might explain why the high-length fragments could not resolve in the amplifications with highest yields.]

“Lower [annealing temperature] enhanced the PS-effect under any condition. [Annealing temperature] greatly affected the PS-effect when the primer concentration was low (P2 = 0.04M). [The] average length of the products was longer at lower [annealing temperature]. [...] From 50 to 61.2°C), longer products (up to 10 kb) wer eefficiently amplified. When [annealing temperature] was higher (>65.9°C), much shorter products (<4 kb) were efficiently amplified and longer products disappeared. [Annealing temperature] also affected the PS-effect when primer concentration was 15-fold higher [...] The average product length between higher (70◦C) and lower (60◦C) [annealing temperature] was significantly different, regardless of adaptors used.”

“[Only] with the longest adaptor [...] we were able to amplify products in a wide range (from 0.25 to 10 kb) simultaneously with a slight over-representation of longer molecules. [To] representatively amplify a complex sample, relatively long ITR (compared to primer length), high [annealing temperature], and high concentratio nof primer should be used.”

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Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing.

Morin R, Bainbridge M, Fejes A, Hirst M, Krzywinski M, Pugh T, McDonald H, Varhol R, Jones S, Marra M.

Biotechniques. 2008 Jul;45(1):81-94. doi:10.2144/000112900

Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing.

Tempate switching and RT with random hexamers, followed by sonication, adapter ligation, size fractionnation, amplification and sequencing.