This page lists the announcements of all publication of our unit, starting from April 1st. For older works, please refer to each people's page.
NanoCAGE: A Method for the Analysis of Coding and Noncoding 5′-Capped Transcriptomes.
We published a major update of the nanoCAGE protocol in an extensive book chapter (53 pages!) in Methods in Molecular Biology volume 1543 (see reference to Poulain et al., at the bottom of this page). The biggest changes, illustrated below, are the introduction of unique molecular identifiers in the 5′ linker, and the replacement of the "library" PCR by a fragmentation and amplification step using the standard "tagmentation" kit from Illumina.
The tagmentation step introduces linkers that prepare for multiplexing and sequencing on the Illumina platform. Whole-genome and whole-transcriptome analysis methods use this kit extensively. To adapt it for nanoCAGE, we replaced one of the amplification primers by a custom primer matching our template-switching oligonucleotide, so that only the 5′ fragments will be amplified.
The book chapter includes a 3-page bench workflow summary (see below), which is also available for download from GitHub.
Poulain S, Kato S, Arnaud O, Morlighem JÉ, Suzuki M, Plessy C✉, Harbers M✉. NanoCAGE: A Method for the Analysis of Coding and Noncoding 5'-Capped Transcriptomes. Methods Mol Biol. 2017 1543:57-109 PubMed: 28349422
Targeted reduction of highly abundant transcripts with pseudo-random primers
In quantitative analysis of gene expression using DNA sequencers, the precision depends on the number of sequence reads, and each of these reads has a cost. Therefore, if one prevents uninformative sequences to be read, the cost/performance of the analysis increases. We have developed a method to deplete target sequences from transcriptome libraries (typically nanoCAGE).
Before sequencing, RNA molecules are usually converted into DNA molecules with an enzyme, the reverse-transcriptase, that uses short DNA oligonucleotides as synthesis primers. Reactions to convert the whole transcriptome are often conduced with a "random" mixture of 4,096 different primers, that covers all the possible combinations of A, C, G and T on 6 consecutive nucleotides. In 2009, Armour and collaborators showed that by using a subset of these "random" primers, one can avoid the conversion of target RNA molecules (typically the ribosomal RNAs, which are highly abundant but carry little information). However, this subset was several hundreds of oligonucleotides, which is costly to synthesize.
Observing that the reverse-transcriptase is highly tolerant to mismatches, we reasoned that a similar result could be obtained with a dramatically lower number of oligonucleotides, which we called "pseudo-random primers". In an article published this month in BioTechniques (Arnaud et al., 2016), we demonstrate our approach by reducing either ribosomal or hemoglobin RNA. Importantly, our method also applies to the reduction of the artifacts created by the cross priming of the oligonucleotide tails.
During the publication process of this work, we also explored the new ways of communicating scientific results on Internet. We deposited our presubmission manuscript to the "bioRxiv" repository, and the scripts of our bioinformatics analysis as supplemental material in GitHub.
Characterization of novel transcripts of human papillomavirus type 16 using CAGE technology
Transcriptome analysis of HPV-infected cells using CAGE and 3′ RACE shows the presence of transcripts on the minus strand of the HPV genome, antisense to the known protein-coding genes.
Our research unit joined this collaboration by providing our protocol for 3′ RACE analysis of non-polyadenylated transcripts. We are now working on the characterisation of these transcripts more in details using high-throughput sequencing of full-length cDNAs.
Taguchi A, Nagasaka K✉, Kawana K✉, Hashimoto K, Kusumoto-Matsuo R, Plessy C, Thomas M, Nakamura H, Bonetti A, Oda K, Kukimoto I, Carninci P, Banks L, Osuga Y, Fujii T. Characterization of novel transcripts of human papillomavirus type 16 using CAGE technology. J Virol. 2015 Feb;89(4):2448-52 PubMed: 25505068
Digital expression profiling of the compartmentalized translatome of Purkinje neurons
In collaboration with Molecular Neurocybernetics Unit at the the RIKEN's Brain Science Institute, we have profiled the translatome of Purkinje neurons, to identify expressed proteins and the non-coding RNAs that may regulate their translation. The press release on RIKEN's website summarises our findings.
In this work, we made an extensive use of the our CAGEscan approach to link promoters to functional transcripts. Since the study was done on rats, in which gene models are not comprehensive in 5′, CAGEscan was instrumental to distinguish main promoters of known genes from novel promoters of unknown genes. This article is the reference use for the CAGEscan clustering tools for which we released the source code in 2013.
Note added on August 6th: see also the analysis of hippocampal dendritic RNA using a similar method, published recently by Ainsley et al.
Kratz A☮, Beguin P☮, Kaneko M, Chimura T, Suzuki AM, Matsunaga A, Kato S, Bertin N, Lassmann T, Vigot R, Carninci P, Plessy C, Launey T. Digital expression profiling of the compartmentalized translatome of Purkinje neurons. Genome Research. 2014 Aug;24(8):1396-410 PubMed: 24904046
Two independent transcription initiation codes overlap on vertebrate core promoters
Single-nucleotide resolution analysis of mRNA sequences with CAGE by the FANTOM project revealed earlier the existence of different ways to initiate the transcription of genes, in particular one called sharp, with a strong tendency to always start at the same nucleotide, and one called broad, where the start nucleotide varies withing a window that can be as large as a hundred of bases. The sequences governing the use of one way or the other are the initiation codes featured in the title.
This new article reconciles these different classes and shows that the same genes can be transcribed in sharp or broad manners in different biological contexts, here, the development of zebrafish embryos. This is explained in more details in the press releases at RIKEN and at the MRC.
The following part describes more in detail the contribution of the Genomics Miniaturization Technology Unit. For the validation of the findings in transgenic animals where the initiation codes were altered, we developed the Single-locus CAGE method. The picture below is the figure 3C in the article.
In brief, the Single-locus CAGE method is a gene-specific version of CAGE. The start position (5′ end) of target RNAs is accurately detected by the CAP Trapper method like in CAGE. However, the RNA is reverse-transcribed with gene-specific primers and not random or oligo-dT primers, and therefore the method guarantees a large number of sequence reads for a high coverage of the transcription start site of interest. Lastly, the experimental procedure is simplified by sequencing 5′ ends directly instead of cleaving tags with a restriction enzyme, and the libraries can easily be multiplexed on benchtop sequencers (Illumina MiSeq), allowing for the parallel study of experimental replicates.
Haberle V☮, Li N☮, Hadzhiev Y, Plessy C, Previti C, Nepal C, Gehrig J, Dong X, Akalin A, Suzuki AM, van Ijcken WF, Armant O, Ferg M, Strähle U, Carninci P, Müller F, Lenhard B. Two independent transcription initiation codes overlap on vertebrate core promoters. Nature. 2014 Mar 20;507(7492):381-5 PubMed: 24531765
Broader coverage of gene bodies with LNA/RNA-hybrid template-switching oligonucleotides
We have compared template-switching oligonucleotides of different chemical nature, namely the standard DNA/RNA hybrids with DNA/LNA and pure DNA alternatives, hoping to increase the efficiency of the nanoCAGE protocol. To our surprise the repartition of the signal obtained with the DNA/LNA and the pure DNA oligonucleotides covered gene bodies more flatly, that is, did not show the same preference for the 5′ end as with DNA/RNA.
We published these results in BMC Genomics, where we discussed the implications for the mechanism of template switching, and the possible uses of the DNA/LNA oligonucleotides. This article also features an extensive source code listing in its supplementary material, which may be useful to people doing pairwise comparisons of triplicated CAGE libraries.
While our article was in revision, another study of LNA-containing oligonucleotides (a DNA/RNA/LNA triple hybrid) was published by Picelli et al.
Harbers M, Kato S, de Hoon M, Hayashizaki Y, Carninci P, Plessy C. Comparison of RNA- or LNA-hybrid oligonucleotides in template-switching reactions for high-speed sequencing library preparation. BMC Genomics. 2013 Sep 30 14(1):665. PubMed: 24079827