title: Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data.
abstract: 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.
authors.affiliation: Bioinformatics Graduate Program, Boston University, Boston, Massachusetts 02215, USA Section of Computational Biomedicine, Boston University Medical Center, Boston, Massachusetts 02118, USA.
authors.forename: Joshua D
authors.identifier: http://orcid.org/0000-0003-0780-8662
authors.initials: JD
authors.lastname: Campbell
authors.affiliation: Section of Computational Biomedicine, Boston University Medical Center, Boston, Massachusetts 02118, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Liu
authors.affiliation: Section of Computational Biomedicine, Boston University Medical Center, Boston, Massachusetts 02118, USA.
authors.forename: Lingqi
authors.initials: L
authors.lastname: Luo
authors.affiliation: Section of Computational Biomedicine, Boston University Medical Center, Boston, Massachusetts 02118, USA.
authors.forename: Ji
authors.initials: J
authors.lastname: Xiao
authors.affiliation: Bioinformatics Graduate Program, Boston University, Boston, Massachusetts 02215, USA Section of Computational Biomedicine, Boston University Medical Center, Boston, Massachusetts 02118, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Gerrein
authors.affiliation: Simmons Center for Interstitial Lung Disease and Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Brenda
authors.initials: B
authors.lastname: Juan-Guardela
authors.affiliation: Simmons Center for Interstitial Lung Disease and Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: John
authors.initials: J
authors.lastname: Tedrow
authors.affiliation: Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
authors.forename: Yuriy O
authors.initials: YO
authors.lastname: Alekseyev
authors.affiliation: Center for Genes, Environment and Health and Department of Medicine, National Jewish Health, Denver, Colorado 80206, USA.
authors.forename: Ivana V
authors.initials: IV
authors.lastname: Yang
authors.affiliation: Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, Massachusetts 02215, USA.
authors.forename: Mick
authors.initials: M
authors.lastname: Correll
authors.affiliation: Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Geraci
authors.affiliation: Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, Massachusetts 02215, USA.
authors.forename: John
authors.initials: J
authors.lastname: Quackenbush
authors.affiliation: Simmons Center for Interstitial Lung Disease and Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Sciurba
authors.affiliation: Center for Genes, Environment and Health and Department of Medicine, National Jewish Health, Denver, Colorado 80206, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Schwartz
authors.affiliation: Simmons Center for Interstitial Lung Disease and Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Naftali
authors.initials: N
authors.lastname: Kaminski
authors.affiliation: Bioinformatics Graduate Program, Boston University, Boston, Massachusetts 02215, USA Section of Computational Biomedicine, Boston University Medical Center, Boston, Massachusetts 02118, USA.
authors.forename: W Evan
authors.initials: WE
authors.lastname: Johnson
authors.affiliation: Bioinformatics Graduate Program, Boston University, Boston, Massachusetts 02215, USA Section of Computational Biomedicine, Boston University Medical Center, Boston, Massachusetts 02118, USA.
authors.forename: Stefano
authors.initials: S
authors.lastname: Monti
authors.affiliation: Bioinformatics Graduate Program, Boston University, Boston, Massachusetts 02215, USA Section of Computational Biomedicine, Boston University Medical Center, Boston, Massachusetts 02118, USA.
authors.forename: Avrum
authors.initials: A
authors.lastname: Spira
authors.affiliation: Section of Computational Biomedicine, Boston University Medical Center, Boston, Massachusetts 02118, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Beane
authors.affiliation: Bioinformatics Graduate Program, Boston University, Boston, Massachusetts 02215, USA Section of Computational Biomedicine, Boston University Medical Center, Boston, Massachusetts 02118, USA Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA mlenburg@bu.edu.
authors.forename: Marc E
authors.initials: ME
authors.lastname: Lenburg
mesh_terms: D020869:Gene Expression Profiling; D006801:Humans; D008168:Lung / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D059467:Transcriptome
chemical_list: D035683:MicroRNAs
keywords: coverage; detection; differential expression; microRNA; multiplexing; sequencing