Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data

Joshua D. Campbell, Gang Liu, Lingqi Luo, Ji Xiao, Joseph Gerrein, Brenda Juan-Guardela, John Tedrow, Yuriy O. Alekseyev, Ivana V. Yang, Mick Correll, Mark Geraci, John Quackenbush, Frank Sciurba, David A. Schwartz, Naftali Kaminski, W. Evan Johnson, Stefano Monti, Avrum Spira, Jennifer Beane, Marc E. Lenburg

Research output: Contribution to journalArticle

16 Scopus citations


Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome. The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However, multiplexing involves a trade-off between increased number of sequenced samples and reduced number of reads per sample (i.e., lower depth of coverage). To assess the effect of different sequencing depths owing to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing one, three, six, nine, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization, replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression >1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the log2 number of reads aligning to microRNA loci (R = 0.96). However, most additional microRNAs detected in samples with greater sequencing depth were in the range of expression which had lower fold change reproducibility. These findings elucidate the trade-off between increasing the number of samples in a multiplex with decreasing sequencing depth and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.

Original languageEnglish (US)
Pages (from-to)164-171
Number of pages8
Issue number2
StatePublished - Feb 1 2015


  • Coverage
  • Detection
  • Differential expression
  • MicroRNA
  • Multiplexing
  • Sequencing

ASJC Scopus subject areas

  • Molecular Biology

Fingerprint Dive into the research topics of 'Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data'. Together they form a unique fingerprint.

  • Cite this

    Campbell, J. D., Liu, G., Luo, L., Xiao, J., Gerrein, J., Juan-Guardela, B., Tedrow, J., Alekseyev, Y. O., Yang, I. V., Correll, M., Geraci, M., Quackenbush, J., Sciurba, F., Schwartz, D. A., Kaminski, N., Johnson, W. E., Monti, S., Spira, A., Beane, J., & Lenburg, M. E. (2015). Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data. RNA, 21(2), 164-171.