Comparison of RNA- or LNA-hybrid oligonucleotides in template-switching reactions for high-speed sequencing library preparation

Harbers, Matthias; Kato, Sachi; Hoon, Michiel de; Hayashizaki, Yoshihide; Carninci, Piero; Plessy, Charles

doi:10.1186/1471-2164-14-665

Cited by 23 publications

(21 citation statements)

References 25 publications

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“…Library preparation and creation of the BAM files containing mapped reads was performed as described above. The mapped CAGEseq reads were grouped into single base pair promoters by determining all positions in the genome to which the 5′ end of at least one CAGE read with mapping quality of at least 20 mapped to and outputting tpm normalized expression values per base pair position [ 33 ].…”

Section: Methodsmentioning

confidence: 99%

C9orf72 is differentially expressed in the central nervous system and myeloid cells and consistently reduced in C9orf72, MAPT and GRN mutation carriers

Rizzu

Blauwendraat

Heetveld

et al. 2016

acta neuropathol commun

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A non-coding hexanucleotide repeat expansion (HRE) in C9orf72 is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) acting through a loss of function mechanism due to haploinsufficiency of C9orf72 or a gain of function mediated by aggregates of bidirectionally transcribed HRE-RNAs translated into di-peptide repeat (DPR) proteins. To fully understand regulation of C9orf72 expression we surveyed the C9orf72 locus using Cap Analysis of Gene Expression sequence data (CAGEseq). We observed C9orf72 was generally lowly expressed with the exception of a subset of myeloid cells, particularly CD14+ monocytes that showed up to seven fold higher expression as compared to central nervous system (CNS) and other tissues. The expression profile at the C9orf72 locus showed a complex architecture with differential expression of the transcription start sites (TSSs) for the annotated C9orf72 transcripts between myeloid and CNS tissues suggesting cell and/or tissue specific functions. We further detected novel TSSs in both the sense and antisense strand at the C9orf72 locus and confirmed their existence in brain tissues and CD14+ monocytes. Interestingly, our experiments showed a consistent decrease of C9orf72 coding transcripts not only in brain tissue and monocytes from C9orf72-HRE patients, but also in brains from MAPT and GRN mutation carriers together with an increase in antisense transcripts suggesting these could play a role in regulation of C9orf72. We found that the non-HRE related expression changes cannot be explained by promoter methylation but by the presence of the C9orf72-HRE risk haplotype and unknown functional interactions between C9orf72, MAPT and GRN.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0306-7) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

C9orf72 is differentially expressed in the central nervous system and myeloid cells and consistently reduced in C9orf72, MAPT and GRN mutation carriers

Rizzu

Blauwendraat

Heetveld

et al. 2016

acta neuropathol commun

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“…This protocol involves the capture of polyadenylated RNA transcripts, followed by template switching at the 3′-end and RACE-PCR amplification of the resulting cDNA (Clontech SMART RACE cDNA Amplification Kit), and then using illumina’s Nextera library preparation kit. This approach preferentially enriches RNAs that undergo posttranscriptional processing, such as polyadenylation and 5′-capping ( Harbers et al 2013 ), such as mRNAs and lncRNAs. Libraries were sequenced on the illumina GAIIx platform in paired end 50 bp mode.…”

Section: Methodsmentioning

confidence: 99%

Developmental Progression in the CoralAcropora digitiferaIs Controlled by Differential Expression of Distinct Regulatory Gene Networks

et al. 2016

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Corals belong to the most basal class of the Phylum Cnidaria, which is considered the sister group of bilaterian animals, and thus have become an emerging model to study the evolution of developmental mechanisms. Although cell renewal, differentiation, and maintenance of pluripotency are cellular events shared by multicellular animals, the cellular basis of these fundamental biological processes are still poorly understood. To understand how changes in gene expression regulate morphogenetic transitions at the base of the eumetazoa, we performed quantitative RNA-seq analysis during Acropora digitifera’s development. We collected embryonic, larval, and adult samples to characterize stage-specific transcription profiles, as well as broad expression patterns. Transcription profiles reconstructed development revealing two main expression clusters. The first cluster grouped blastula and gastrula and the second grouped subsequent developmental time points. Consistently, we observed clear differences in gene expression between early and late developmental transitions, with higher numbers of differentially expressed genes and fold changes around gastrulation. Furthermore, we identified three coexpression clusters that represented discrete gene expression patterns. During early transitions, transcriptional networks seemed to regulate cellular fate and morphogenesis of the larval body. In late transitions, these networks seemed to play important roles preparing planulae for switch in lifestyle and regulation of adult processes. Although developmental progression in A. digitifera is regulated to some extent by differential coexpression of well-defined gene networks, stage-specific transcription profiles appear to be independent entities. While negative regulation of transcription is predominant in early development, cell differentiation was upregulated in larval and adult stages.

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“…The length of the associated 5' UTR was only 3 bp. It was unlikely to be the result of RNA degradation during library preparation, because the 5'cap is required for a successful template switch when using 5'RACE protocol [40]. Moreover, we found the 5' UTR for IGHV1-17*02 in 6 subjects, a strong evidence for its authenticity.…”

Section: Discussionmentioning

confidence: 66%

Antibody Upstream Sequence Diversity and Its Biological Implications Revealed by Repertoire Sequencing

Zhu

Yang

et al. 2020

Preprint

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The sequence upstream of antibody variable region (Antibody Upstream Sequence, or AUS) consists of 5’ untranslated region (5’ UTR) and two leader regions, L-PART1 and L-PART2. The sequence variations in AUS affect the efficiency of PCR amplification, mRNA translation, and subsequent PCR-based antibody quantification as well as antibody engineering. Despite their importance, the diversity of AUSs has long been neglected. Utilizing the rapid amplification of cDNA ends (5’RACE) and high-throughput antibody repertoire sequencing (Rep-Seq) technique, we acquired full-length AUSs for human, rhesus macaque (RM), cynomolgus macaque (CM), mouse, and rat. We designed a bioinformatics pipeline and discovered 2,957 unique AUSs, corresponding to 2,786 and 1,159 unique sequences for 5’ UTR and leader, respectively. Comparing with the leader records in the international ImMunoGeneTics (IMGT), while 529 were identical, 313 were with single nucleotide polymorphisms (SNPs), 280 were totally new, and 37 updated the incomplete records. The diversity of AUSs’ impact on related antibody biology was also probed. Taken together, our findings would facilitate Rep-Seq primer design for capturing antibodies comprehensively and efficiently as well as provide a valuable resource for antibody engineering and the studies of antibody at the molecular level.

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Comparison of RNA- or LNA-hybrid oligonucleotides in template-switching reactions for high-speed sequencing library preparation

Cited by 23 publications

References 25 publications

C9orf72 is differentially expressed in the central nervous system and myeloid cells and consistently reduced in C9orf72, MAPT and GRN mutation carriers

C9orf72 is differentially expressed in the central nervous system and myeloid cells and consistently reduced in C9orf72, MAPT and GRN mutation carriers

Developmental Progression in the CoralAcropora digitiferaIs Controlled by Differential Expression of Distinct Regulatory Gene Networks

Antibody Upstream Sequence Diversity and Its Biological Implications Revealed by Repertoire Sequencing

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