TY - JOUR
T1 - IsoSeek for unbiased and UMI-informed sequencing of miRNAs from low input samples at single-nucleotide resolution
AU - van Eijndhoven, Monique A. J.
AU - Scheepbouwer, Chantal
AU - Aparicio-Puerta, Ernesto
AU - Hackenberg, Michael
AU - Pegtel, D. Michiel
N1 - Funding Information: This work was supported by multiple grants awarded to D.M.P. including NWO Perspectief Cancer-ID, TKI-Health Holland AQrate, and Cancer Center Amsterdam Foundation. We thank E.J. Bradley for technical advice. Figure 3 and part of the graphical abstract were created with BioRender.com. Conceptualization, M.A.J.v.E. and D.M.P.; methodology, M.A.J.v.E. M.H. and D.M.P.; investigation, M.A.J.v.E.; writing - initial draft, M.A.J.v.E. and D.M.P.; reviewing and editing, M.A.J.v.E. C.S. E.A.-P. M.H. and D.M.P.; technical advice, C.S.; data analysis, M.A.J.v.E. E.A.-P. and M.H.; funding acquisition, D.M.P. D.M.P. and M.H. are co-founders of ExBiome BV and D.M.P. is CSO of ExBiome BV. D.M.P. and M.H. are inventors on a patent related to this work (WO2015165779A3). M.A.J.v.E. received travel compensation from QIAGEN. Funding Information: This work was supported by multiple grants awarded to D.M.P. including NWO Perspectief Cancer-ID, TKI -Health Holland AQrate, and Cancer Center Amsterdam Foundation. We thank E.J. Bradley for technical advice. Figure 3 and part of the graphical abstract were created with BioRender.com . Publisher Copyright: © 2023 The Authors
PY - 2023/12/15
Y1 - 2023/12/15
N2 - Besides canonical microRNAs (miRNAs), sequence-based variations called isomiRs have biological relevance and diagnostic potential; however, accurate calling of these post-transcriptional modifications is challenging, especially for low input samples. Here, we present IsoSeek, a sequencing protocol that reduces ligation and PCR amplification bias and improves the accuracy of miRNA detection in low input samples. We describe steps for using randomized adapters combined with unique molecular identifiers (UMI), library quantification, and sequencing, followed by detailed procedures for data processing and analysis. For complete details on the use and execution of this protocol, please refer to C. Gómez-Martín et al. (2023) 1 and Van Eijndhoven et al. (2021).
AB - Besides canonical microRNAs (miRNAs), sequence-based variations called isomiRs have biological relevance and diagnostic potential; however, accurate calling of these post-transcriptional modifications is challenging, especially for low input samples. Here, we present IsoSeek, a sequencing protocol that reduces ligation and PCR amplification bias and improves the accuracy of miRNA detection in low input samples. We describe steps for using randomized adapters combined with unique molecular identifiers (UMI), library quantification, and sequencing, followed by detailed procedures for data processing and analysis. For complete details on the use and execution of this protocol, please refer to C. Gómez-Martín et al. (2023) 1 and Van Eijndhoven et al. (2021).
KW - Bioinformatics
KW - Biotechnology and Bioengineering
KW - Genetics
KW - Genomics
UR - http://www.scopus.com/inward/record.url?scp=85174160090&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.xpro.2023.102645
DO - https://doi.org/10.1016/j.xpro.2023.102645
M3 - Article
C2 - 37858475
SN - 2666-1667
VL - 4
JO - STAR Protocols
JF - STAR Protocols
IS - 4
M1 - 102645
ER -