Name: identification of circular rnas using rna sequencing
Uploaded: 2019-11-14T11:00:00
Description: Watch this Scientific Journal Video about Identification of Circular RNAs using RNA Sequencing at JoVE.com

Vi er taknemmelige for banneret Sun Health Research Institute Brain og Body donation program (BBDP) af Sun City, Arizona for levering af menneskelige hjernevæv. BBDP er blevet støttet af det nationale Institut for neurologiske lidelser og slagtilfælde (U24 NS072026 national hjerne-og vævs ressource for Parkinsons sygdom og beslægtede lidelser), National Institute on Aging (P30AG19610 Arizona Alzheimers sygdom Core Center), Arizona Department of Health Services (kontrakt 211002, Arizona Alzheimers Research Center), Arizona biomedicinsk forskning provision (kontrakter 4001, 0011, 05-901 og 1001 til Arizona Parkinsons sygdom konsortium) og Michael Jørgensen. Fox Foundation for Parkinsons forskning27. Denne undersøgelse blev også støttet af DHS og staten Arizona (ADHS Grant # ADHS14-052688). Vi takker også Andrea Schmitt (banner Research) og Cynthia Lechuga (TGen) for administrativ støtte.

Denne forskning er blevet udført i overensstemmelse med alle institutionelle, nationale og internationale retningslinjer for menneskers velfærd. Hjernevæv blev opnået fra banneret Sun Health Research Institute Brain og Body donation program i Sun City, AZ. Operationerne i hjernen og organ donation program er godkendt af den vestlige institutionelle Review Board (WIRB Protocol #20120821). Alle eller deres juridiske repræsentanter underskrev det informerede samtykke. Kommercielle (ikke-hjerne) bioprøver blev købt ho...

<ol>
	<li>Salzman, J., Gawad, C., Wang, P. L., Lacayo, N., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNAs+are+the+predominant+transcript+isoform+from+hundreds+of+human+genes+in+diverse+cell+types.">Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types.</a> PloS One. 7 (2), e30733 (2012).</li><li>Jeck, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNAs+are+abundant,+conserved,+and+associated+with+ALU+repeats.">Circular RNAs are abundant, conserved, and associated with ALU repeats.</a> RNA. 19 (2), 141-157 (2013).</li><li>Memczak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNAs+are+a+large+class+of+animal+RNAs+with+regulatory+potency.">Circular RNAs are a large class of animal RNAs with regulatory potency.</a> Nature. 495 (7441), 333-338 (2013).</li><li>Sanger, H. L., Klotz, G., Riesner, D., Gross, H. J., Kleinschmidt, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viroids+are+single-stranded+covalently+closed+circular+RNA+molecules+existing+as+highly+base-paired+rod-like+structures.">Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures.</a> Proceedings of the National Academy of Sciences of the United States of America. 73 (11), 3852-3856 (1976).</li><li>Nigro, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrambled+exons.">Scrambled exons.</a> Cell. 64 (3), 607-613 (1991).</li><li>Salzman, J., Chen, R. E., Olsen, M. N., Wang, P. L., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type+specific+features+of+circular+RNA+expression.">Cell-type specific features of circular RNA expression.</a> PLoS Genetics. 9 (9), e1003777 (2013).</li><li>Du, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxo3+circular+RNA+promotes+cardiac+senescence+by+modulating+multiple+factors+associated+with+stress+and+senescence+responses.">Foxo3 circular RNA promotes cardiac senescence by modulating multiple factors associated with stress and senescence responses.</a> European Heart Journal. 38 (18), 1402-1412 (2016).</li><li>Capel, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+transcripts+of+the+testis-determining+gene+Sry+in+adult+mouse+testis.">Circular transcripts of the testis-determining gene Sry in adult mouse testis.</a> Cell. 73 (5), 1019-1030 (1993).</li><li>Hansen, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNA-dependent+gene+silencing+involving+Ago2-mediated+cleavage+of+a+circular+antisense+RNA.">miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA.</a> The EMBO Journal. 30 (21), 4414-4422 (2011).</li><li>Hansen, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+RNA+circles+function+as+efficient+microRNA+sponges.">Natural RNA circles function as efficient microRNA sponges.</a> Nature. 495 (7441), 384-388 (2013).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exon-intron+circular+RNAs+regulate+transcription+in+the+nucleus.">Exon-intron circular RNAs regulate transcription in the nucleus.</a> Nature Structural &amp; Molecular Biology. 22 (3), 256-264 (2015).</li><li>Tan, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+landscape+of+circular+RNA+expression+in+the+human+heart.">A landscape of circular RNA expression in the human heart.</a> Cardiovascular Research. 113 (3), 298-309 (2016).</li><li>Zhong, Z., Lv, M., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+differential+circular+RNA+expression+profiles+reveals+the+regulatory+role+of+circTCF25-miR-103a-3p/miR-107-CDK6+pathway+in+bladder+carcinoma.">Screening differential circular RNA expression profiles reveals the regulatory role of circTCF25-miR-103a-3p/miR-107-CDK6 pathway in bladder carcinoma.</a> Scientific Reports. 6, 30919 (2016).</li><li>Gao, Y., Wang, J., Zhao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CIRI:+an+efficient+and+unbiased+algorithm+for+de+novo+circular+RNA+identification.">CIRI: an efficient and unbiased algorithm for de novo circular RNA identification.</a> Genome Biology. 16, 4-014-0571-0573 (2015).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MapSplice:+accurate+mapping+of+RNA-seq+reads+for+splice+junction+discovery.">MapSplice: accurate mapping of RNA-seq reads for splice junction discovery.</a> Nucleic Acids Research. 38 (18), e178 (2010).</li><li>Szabo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistically+based+splicing+detection+reveals+neural+enrichment+and+tissue-specific+induction+of+circular+RNA+during+human+fetal+development.">Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development.</a> Genome Biology. 16, 126-015-0690-0695 (2015).</li><li>Cheng, J., Metge, F., Dieterich, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+identification+and+quantification+of+circular+RNAs+from+sequencing+data.">Specific identification and quantification of circular RNAs from sequencing data.</a> Bioinformatics. 32 (7), 1094-1096 (2016).</li><li>Zhang, X. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+sequence-mediated+exon+circularization.">Complementary sequence-mediated exon circularization.</a> Cell. 159 (1), 134-147 (2014).</li><li>Rybak-Wolf, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNAs+in+the+mammalian+brain+are+highly+abundant,+conserved,+and+dynamically+expressed.">Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed.</a> Molecular Cell. 58 (5), 870-885 (2015).</li><li>Ji, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+Expression+Landscape+and+Prioritization+of+Circular+RNAs+in+Mammals.">Expanded Expression Landscape and Prioritization of Circular RNAs in Mammals.</a> Cell Reports. 26 (12), 3444-3460 (2019).</li><li>Hansen, T. B., Ven&#248;, M. T., Damgaard, C. K., Kjems, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+circular+RNA+prediction+tools.">Comparison of circular RNA prediction tools.</a> Nucleic Acids Research. 44 (6), e58 (2016).</li><li>Zeng, X., Lin, W., Guo, M., Zou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+overview+and+evaluation+of+circular+RNA+detection+tools.">A comprehensive overview and evaluation of circular RNA detection tools.</a> PLoS Comput Biol. 13 (6), e1005420 (2017).</li><li>Sekar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACValidator:+a+novel+assembly-based+approach+for+in+silico+validation+of+circular+RNAs.">ACValidator: a novel assembly-based approach for in silico validation of circular RNAs.</a> bioRxiv. , (2019).</li><li>Westholm, J. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+drosophila+circular+RNAs+reveals+their+structural+and+sequence+properties+and+age-dependent+neural+accumulation.">Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation.</a> Cell Reports. 9 (5), 1966-1980 (2014).</li><li>Szabo, L., Salzman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+circular+RNAs:+bioinformatic+and+experimental+challenges.">Detecting circular RNAs: bioinformatic and experimental challenges.</a> Nature Reviews Genetics. 17 (11), 679-692 (2016).</li><li>Zheng, Y., Ji, P., Chen, S., Hou, L., Zhao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+full-length+circular+RNAs+enables+isoform-level+quantification.">Reconstruction of full-length circular RNAs enables isoform-level quantification.</a> Genome Medicine. 11 (1), 2 (2019).</li><li>Beach, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arizona+study+of+aging+and+neurodegenerative+disorders+and+brain+and+body+donation+program.">Arizona study of aging and neurodegenerative disorders and brain and body donation program.</a> Neuropathology. 35 (4), 354-389 (2015).</li></ol>

I denne undersøgelse blev to kommercielt tilgængelige Biblioteks forberedelses kits, præ-behandlingsmuligheder og input-RNA-beløb testet for at optimere en circRNA-berigelses protokol til opførelse af circRNA sekvensering biblioteker. På baggrund af undersøgelsens vurderinger er en række vigtige aspekter og kritiske trin i skabelsen af circRNA-sekvensering af biblioteker tydeligt. Vores evaluering bekræfter nytten af RNase R forbehandling, som afspejlet i det øgede antal circRNAs detekteret. Samlet set blev der...

Cirkulære RNAs (circrnas) er endogene, ikke-kodning RNAs, der har fået opmærksomhed i betragtning af deres omsiggribende udtryk i eukaryote transkriptomet1,2,3. De er dannet, når exons back-Splice til hinanden og dermed oprindeligt blev anset for at være splejsning artefakter4,5. Men, nylige undersøgelser har vist, at circrnas udviser celletype, væv, og udviklingsmæssige fase specifikke udtryk3,6 og er evolutionært bevaret2,3. Desuden er de involveret i mægling af protein-protein interaktioner7, mikro-RNA (Mirna) bindende3,8,9,10, og regulering af forældrenes gen transkriptionen11.
I klassisk RNA-sekvensering (RNA-SEQ), kan circRNAs være helt tabt under biblioteks konstruktionen som et resultat af poly-et valg for mRNAs eller kan være vanskeligt at isolere i betragtning af deres lave overflod. Men, nylige circrna karakterisering undersøgelser har indarbejdet en præ-behandling trin ved hjælp af RNase Rasmussen for at berige for circrnas2,12,13. RNase Rasmussen er en exoribonuclease, der fordøjer lineære RNAs, efterlader cirkulære RNA strukturer. CircRNA-berigelses protokoller blev optimeret ved at generere og sammenligne data fra to kommercielt tilgængelige hele transkriptome Library byggesæt, med og uden et RNase R forbehandlings trin, og ved hjælp af varierende mængder af samlet RNA-input (1 til 4 μg). Den optimerede protokol blev derefter brugt til at evaluere den overflod af circRNAs på tværs af fem forskellige hjerneområder (cerebellum [BC], ringere parietal lap [IP], Middle Temporal gyrus [MG], occipital cortex [OC] og overlegne frontal gyrus [SF]) og fire andre vævstyper (leveren [LV], lunge [LU], lymfeknude [LN] og bugspytkirtel [PA]). RNA-SEQ-bibliotekerne blev parret sekvenserede, og data blev analyseret ved hjælp af seks forskellige circRNA-forudsigelses algoritmer: find_circ3, Ciri14, mapsplice15, Knife16, DCC17og circexplorer18. Baseret på vores analyse, det højeste antal af unikke circRNAs blev detekteret, når du bruger en strandede samlede RNA bibliotek forberedelse kit med RNase R forbehandling og 4 μg samlede input RNA. Den optimerede protokol er beskrevet her. Som tidligere rapporteret19,20, den højeste berigelse af circrnas blev observeret i hjernen sammenlignet med andre vævstyper.

<table>
	<tbody>
		<tr>
			<td>1000 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>GP-L1000F</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>SR L 10F</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>SR L 200F</td>
			<td></td>
		</tr>
		<tr>
			<td>2200 TapeStation Accessories (foil covers)</td>
			<td>Agilent Technologies</td>
			<td>5067-5154</td>
			<td></td>
		</tr>
		<tr>
			<td>2200 TapeStation Accessories (tips)</td>
			<td>Agilent Technologies</td>
			<td>5067-5153</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Film for Microplates</td>
			<td>VWR</td>
			<td>60941-064</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure XP Beads 450 mL</td>
			<td>Beckman Coulter</td>
			<td>A63882</td>
			<td>PCR purification</td>
		</tr>
		<tr>
			<td>Eppendorf twin.tec 96-Well PCR Plates</td>
			<td>VWR</td>
			<td>951020401</td>
			<td></td>
		</tr>
		<tr>
			<td>High Sensitivity D1000 reagents</td>
			<td>Agilent Technologies</td>
			<td>5067-5585</td>
			<td></td>
		</tr>
		<tr>
			<td>High Sensitivity D1000 ScreenTape</td>
			<td>Agilent Technologies</td>
			<td>5067-5584</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 2500 Sequencing System</td>
			<td>Illumina</td>
			<td>SY-401-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 3000/4000 PE Cluster Kit</td>
			<td>Illumina</td>
			<td>PE-410-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 3000/4000 SBS Kit (150 cycles)</td>
			<td>Illumina</td>
			<td>FC-410-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 4000 Sequencing System</td>
			<td>Illumina</td>
			<td>SY-401-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq PE PE Rapid Cluster Kit v2</td>
			<td>Illumina</td>
			<td>PE-402-4002</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq Rapid SBS Kit v2 (50 cycle)</td>
			<td>Illumina</td>
			<td>FC-402-4022</td>
			<td></td>
		</tr>
		<tr>
			<td>Kapa Total RNA Kit</td>
			<td>Roche</td>
			<td>KK8400</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular biology grade ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP28184</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Assay Tubes</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA High Sense Assay Kit</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>Q32854</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA cleanup and concentrator - 5</td>
			<td>Zymo</td>
			<td>RCC-100</td>
			<td>Contains purification columns, collection tubes</td>
		</tr>
		<tr>
			<td>RNAClean XP beads</td>
			<td>Beckman Coulter Genomics</td>
			<td></td>
			<td>RNA Cleanup beads</td>
		</tr>
		<tr>
			<td>Rnase R</td>
			<td>Lucigen</td>
			<td>RNR07250</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase 10,000 units</td>
			<td>ThermoFisher (LifeTech)</td>
			<td>18064014</td>
			<td></td>
		</tr>
		<tr>
			<td>TapeStation 2200</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>Nucleic Acid analyzer</td>
		</tr>
		<tr>
			<td>TElowE</td>
			<td>VWR</td>
			<td>10128-588</td>
			<td></td>
		</tr>
		<tr>
			<td>TruSeq Stranded Total RNA Library Prep Kit</td>
			<td>Illumina</td>
			<td>20020596</td>
			<td>Kit used in section 3</td>
		</tr>
		<tr>
			<td>Two-Compartment Divided Tray</td>
			<td>VWR</td>
			<td>3054-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Water</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal control RNA</td>
			<td>Agilent</td>
			<td>740000</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of circular rnas using rna sequencing

Cirkulære RNAs (circRNAs) er en klasse af ikke-kodning RNAs involveret i funktioner, herunder mikro-RNA (miRNA) regulering, mægling af protein-protein interaktioner, og regulering af forældrenes gen transkriptionen. I klassisk næste generation af RNA-sekvensering (RNA-SEQ), er circRNAs typisk overset som et resultat af poly-A udvælgelse under opførelsen af mRNA biblioteker, eller findes på meget lav overflod, og er derfor vanskeligt at isolere og opdage. Her blev en circRNA Library Construction Protocol optimeret ved at sammenligne Biblioteks forberedelses kits, forbehandlings muligheder og forskellige samlede RNA-input beløb. To kommercielt tilgængelige hele transcriptome Library Preparation kits, med og uden RNase R forbehandling, og ved hjælp af variable mængder af samlede RNA-input (1 til 4 μg), blev testet. Endelig, flere vævstyper; herunder lever, lunge, lymfeknude, og bugspytkirtel; samt flere hjerneområder; herunder cerebellum, ringere parietal lap, Middle Temporal gyrus, occipital cortex, og overlegne frontal gyrus; blev sammenlignet med at evaluere circRNA overflod på tværs af vævstyper. Analyse af de genererede RNA-SEQ-data ved hjælp af seks forskellige circRNA-detektionsværktøjer (find_circ, CIRI, Mapsplice, KNIFE, DCC og CIRCexplorer) afslørede, at et strandede total RNA-Biblioteks forberedelses kit med RNase R-forbehandling og 4 μg RNA-input er den optimale metode til identifikation af det højeste relative antal circRNAs. I overensstemmelse med tidligere fund blev den højeste berigelse af circRNAs observeret i hjernevæv sammenlignet med andre vævstyper.

Data genereret ved hjælp af en kommercielt tilgængelig Universal Control RNA (UC) og ved hjælp af to bibliotek forberedelse kits, som begge omfatter en Ribo-udtynding trin i deres protokoller, blev først vurderet. Ved hjælp af en analytisk arbejdsproces (workflow for data analyse, afsnit 4) blev der samlet set fundet et højere antal circRNAs i data sætternes TruSeq-datasæt sammenlignet med kapa-dem (figur 1). Selv om ribosomale RNA (rRNA)-procenterne var under 5% i datasæt fra begge...

Watch this Scientific Journal Video about Identification of Circular RNAs using RNA Sequencing at JoVE.com

Identification of Circular RNAs using RNA Sequencing

Cirkulære RNAs (circRNAs) er ikke-kodning RNAs, der kan have roller i transkriptional regulering og medierende interaktioner mellem proteiner. Efter vurdering af forskellige parametre for opførelse af circRNA sekvensering biblioteker, en protokol blev udarbejdet ved hjælp af strandede samlede RNA bibliotek forberedelse med RNase R præ-behandling og præsenteres her.

Identifikation af cirkulære RNAs ved hjælp af RNA-sekvensering

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Denne protokol er resultatet af at evaluere centrale aspekter af circRNA bibliotek forberedelse, såsom kit type, RNase R behandling, input mængder og deres indvirkning på circRNA afsløring. Det muliggør optimal circRNA-detektion og giver data om justerbare parametre afhængigt af brugerens behov. Identifikation circRNAs i forskellige prøvetyper vil hjælpe os til bedre at forstå fordelingen af deres udtryk og sætter scenen for delineating den funktionelle rolle af disse molekyler.Denne metode kan anvendes på enhver total RNA-prøve. Det er vigtigt at opbevare RNA-prøver på is, før protokollen påbegyndes. Vurder RIN- og DV200-værdierne på Agilent Bioanalyzer eller TapeStation før forberedelsen, og følg de relevante procedurer, når du arbejder med RNA.Begynd med at fortynde det samlede RNA til fire mikrogram i 39 mikroliter RNase-frit vand og pipettere det op og ned for at blande. I et separat rør anvendes 1x RNase R Buffer til at fortynde RNase R til en arbejdskoncentration på to enheder pr. mikroliter. Pipette 39 mikroliter af det samlede RNA og fem mikroliter af 10x RNase R reaktionsbuffer i et 1,5 mikroliters reaktionsrør og blandes.Der tilsættes seks mikroliter af den fortyndede RNase R.Juster derefter pipetten til fuld reaktionsvolumen, og bland opløsningen ved at pipettere op og ned 10 gange. Placer røret i et 37-graders Celsius-vandbad i 10 minutter, og sørg for, at den fulde reaktionsvolumen er nedsænket i vandbadet. Derefter placere røret på is, og straks gå videre med RNA oprydning og koncentration.Før du starter, forberedes RNA-vaskebufferen ved at blande koncentratet med 48 milliliter 100% ethanol, og rensningssøjlerne anbringes i opsamlingsrør. Når du er klar, skal du tilføje to bind RNA-bindingsbuffer til den RNase R-behandlede prøve og blandes godt. Der tilsættes 150 mikroliter på 100% ethanol til blandingen for i alt 300 mikroliter, overfør hele volumen til kolonnen og centrifuge i 30 sekunder.Fjern flowet igennem, og tilføj 400 mikroliter RNA Prep Buffer direkte til kolonnen. Centrifuge i 30 sekunder, og kassér strømmen igennem. Derefter tilsættes 700 mikroliter RNA-vaskebuffer til kolonnen, centrifuge i 30 sekunder, og kassér strømmen igennem.Tilsæt yderligere 400 mikroliter af vaskebufferen, centrifuge i to minutter, og overfør kolonnen til et frisk RNase-frit 1,5 milliliterrør. Tilsæt forsigtigt 11 mikroliter RNase-frit vand direkte til kolonnen ved at holde pipettespidsen lige over kolonnefilteret. Lad prøven sidde i et minut ved stuetemperatur, og derefter centrifuge det i et minut.Sørg for, at der er flow igennem i 1,5-milliliter røret, og kassér kolonnen. Den rensede RNA-prøve kan opbevares ved minus 80 grader Celsius eller anvendes straks til biblioteksforberedelse. Overfør 10 mikroliter af det rensede totale RNA til en ren brønd i en 96-brønd PCR-plade.Der tilsættes fem mikroliter rRNA-bindingsbuffer efterfulgt af fem mikroliter rRNA-fjernelsesblanding, hvortil pipetters forsigtigt op og ned for at blande reagenserne. Forsegle pladen, og inkubere den i fem minutter ved 68 grader Celsius på en forprogrammeret og forvarmet termocykator blok. Efter inkubationen skal pladen sidde ved stuetemperatur i et minut.Fjern forseglingen fra pladen, og tilsæt 35 mikroliter af vortexed rumtemperatur rRNA fjernelse perler til prøven. Juster pipetten til 45 mikroliter, og pipette op og ned 10 til 20 gange for grundigt at blande. Lad pladen sidde ved stuetemperatur i et minut.Overfør pladen til et magnetisk stativ, og inkuber den der i et minut, eller indtil opløsningen er klar. Overfør supernatanten til ny brønd på pladen. Overfør derefter pladen fra den magnetiske stativ til pladestanderen.Vortex RNA oprydning perler, indtil de er godt spredt, og tilsæt 99 mikroliter af perler til prøven. Pipette op og ned for at blande, og inkubere pladen ved stuetemperatur i 10 minutter. Overfør pladen til den magnetiske stativ, og lad den stå der i fem minutter, eller indtil opløsningen ryddes, fjern og kassér derefter al supernatant fra brønden.Med pladen stadig på den magnetiske stativ, tilsættes 200 mikroliter frisklavet 80% ethanol til brønden uden at forstyrre perlerne. Lad ethanolen være i brønden i 30 sekunder, og fjern den og kassér den derefter. Tilføj 11 mikroliter af Elution Buffer til brønden, og pipette det op og ned for at blande.Inkuber pladen ved stuetemperatur i to minutter, og overfør den derefter tilbage til den magnetiske stativ, og hold den der, indtil opløsningen er klar. Overfør 8,5 mikroliter af supernatanten til en ny brønd på samme plade, og tilsæt 8,5 mikroliter af Elute, Primer, Fragment High Mix til hver brønd, der indeholder en prøve. Pipette op og ned 10 gange for at blande grundigt, forsegle pladen, og inkubere den i otte minutter i en thermocycler forvarmet til 94 grader Celsius.Prøven afkøles til fire grader Celsius, pladen fjernes fra termocyren, og den centrifugeres kortvarigt. To biblioteksforberedelsessæt, TruSeq og KAPA, blev vurderet for denne protokol. Samlet set blev der fundet et højere antal cirkulære RNA'er og lavere procentdel af ribosomal RNA ved brug af TruSeq-sættet, så TruSeq blev udvalgt til yderligere eksperimenter.Betydningen af RNase R-forbehandling blev evalueret ved at sammenligne data fra forbehandlede og ikke-forbehandlede biblioteker. Som forventet blev et større antal cirkulære RNA'er konsekvent identificeret i de forbehandlede biblioteker sammenlignet med ikke-forbehandlede biblioteker. Mængden af input RNA blev optimeret til at opdage en højere mangfoldighed af cirkulære RNA'er.Biblioteker blev udarbejdet ved hjælp af et, to og fire mikrogram input RNA, og den højeste mangfoldighed af cirkulære RNA-arter blev observeret ved brug af fire mikrogram total RNA. Den optimerede protokol blev brugt til at sammenligne cirkulære RNA-forekomster i fem hjerneregioner fra fire raske donorer og for andre vævstyper fra seks raske donorer. Samlet set blev der observeret en højere forekomst af cirkulær RNase i hjernen sammenlignet med andre vævstyper.Det er vigtigt at huske at følge passende procedurer, når du arbejder med RNA, herunder iført handsker, grundig rengøring af bænken og pipetter med RNase klude eller spray før du begynder protokollen, og holde RNA på is på forhånd. Efter denne procedure kan der udføres ortogonal validering såsom Sanger-sekvensering af identificerede circRNA-vejkryds. Opdagelsen af nye circRNAs og yderligere beviser for deres tilstedeværelse udskærer et nyt forskningsområde for at udforske deres biologiske funktioner.

identification-of-circular-rnas-using-rna-sequencing

Research

JoVE Journal

Genetics

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Kortlægning af RNA-RNA-interaktioner globalt ved anvendelse af biotinyleret psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

Metaboliske mærkning og profilering af overførsel RNA'er ved hjælp af Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

Retrospektiv mikroRNA sekventering: Supplerende DNA bibliotek forberedelse protokollen ved hjælp af Formalin-fast Paraffin-embedded RNA prøver

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Sjælden begivenhed registrering ved hjælp af fejl-korrigeret DNA og RNA sekventering

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

Høj overførselshastighed identifikation af regulerende gensekvenser ved hjælp af næste generations sekventering af cirkulære kromosom kropsbygning Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.

Overekspression længe Noncoding RNA'er ved hjælp af gen-aktivering CRISPR

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sekvensering af mRNA fra fuldblod ved hjælp af Nanopmalm sekvensering

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Enkelt celle-RNA-sekvensering og analyse af humane bugspytkirtel-øer

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

Identifikation af fodspor af RNA: protein komplekser via RNA Immunopræcipitation i tandem efterfulgt af sekventering (RIPiT-SEQ)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

Improving Small RNA-seq: Less Bias and Better Detection of 2&#039;-O-Methyl RNAs

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

Forbedring af små RNA-SEQ: mindre bias og bedre påvisning af 2 '-O-methyl-RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...