title: Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing.
abstract: BACKGROUND
  Small ncRNAs (sncRNAs) offer great hope as biomarkers of disease and response to treatment. This has been highlighted in the context of several medical conditions such as cancer, liver disease, cardiovascular disease, and central nervous system disorders, among many others. Here we assessed several steps involved in the development of an ncRNA biomarker discovery pipeline, ranging from sample preparation to bioinformatic processing of small RNA sequencing data.
  
  
  METHODS
  A total of 45 biological samples were included in the present study. All libraries were prepared using the Illumina TruSeq Small RNA protocol and sequenced using the HiSeq2500 or MiSeq Illumina sequencers. Small RNA sequencing data was validated using qRT-PCR. At each stage, we evaluated the pros and cons of different techniques that may be suitable for different experimental designs. Evaluation methods included quality of data output in relation to hands-on laboratory time, cost, and efficiency of processing.
  
  
  RESULTS
  Our results show that good quality sequencing libraries can be prepared from small amounts of total RNA and that varying degradation levels in the samples do not have a significant effect on the overall quantification of sncRNAs via NGS. In addition, we describe the strengths and limitations of three commercially available library preparation methods: (1) Novex TBE PAGE gel; (2) Pippin Prep automated gel system; and (3) AMPure XP beads. We describe our bioinformatics pipeline, provide recommendations for sequencing coverage, and describe in detail the expression and distribution of all sncRNAs in four human tissues: whole-blood, brain, heart and liver.
  
  
  CONCLUSIONS
  Ultimately this study provides tools and outcome metrics that will aid researchers and clinicians in choosing an appropriate and effective high-throughput sequencing quantification method for various study designs, and overall generating valuable information that can contribute to our understanding of small ncRNAs as potential biomarkers and mediators of biological functions and disease.
authors.affiliation: McGill Group for Suicide Studies (MGSS), Douglas Mental Health University Institute, McGill University, Frank B Common Pavilion, Room F-2101.2, 6875 LaSalle Boulevard, Montreal, QC, H4H 1R3, Canada. juan.lopez@mail.mcgill.ca.|Department of Human Genetics, McGill University, Montreal, QC, Canada. juan.lopez@mail.mcgill.ca.
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Lopez
authors.affiliation: McGill Group for Suicide Studies (MGSS), Douglas Mental Health University Institute, McGill University, Frank B Common Pavilion, Room F-2101.2, 6875 LaSalle Boulevard, Montreal, QC, H4H 1R3, Canada. alpha.diallo@douglas.mcgill.ca.
authors.forename: Alpha
authors.initials: A
authors.lastname: Diallo
authors.affiliation: McGill Group for Suicide Studies (MGSS), Douglas Mental Health University Institute, McGill University, Frank B Common Pavilion, Room F-2101.2, 6875 LaSalle Boulevard, Montreal, QC, H4H 1R3, Canada. cristiana.cruceanu@mail.mcgill.ca.|Department of Human Genetics, McGill University, Montreal, QC, Canada. cristiana.cruceanu@mail.mcgill.ca.
authors.forename: Cristiana
authors.initials: C
authors.lastname: Cruceanu
authors.affiliation: McGill Group for Suicide Studies (MGSS), Douglas Mental Health University Institute, McGill University, Frank B Common Pavilion, Room F-2101.2, 6875 LaSalle Boulevard, Montreal, QC, H4H 1R3, Canada. laura.fiori@mail.mcgill.ca.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Fiori
authors.affiliation: McGill University and Genome Quebec Innovation Centre, Montreal, QC, Canada. sylvie.laboissiere@mail.mcgill.ca.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Laboissiere
authors.affiliation: McGill University and Genome Quebec Innovation Centre, Montreal, QC, Canada. isabelle.guillet2@mail.mcgill.ca.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Guillet
authors.affiliation: McGill University and Genome Quebec Innovation Centre, Montreal, QC, Canada. joelle.fontaine@mail.mcgill.ca.
authors.forename: Joelle
authors.initials: J
authors.lastname: Fontaine
authors.affiliation: Department of Human Genetics, McGill University, Montreal, QC, Canada. ioannis.ragoussis@mcgill.ca.|McGill University and Genome Quebec Innovation Centre, Montreal, QC, Canada. ioannis.ragoussis@mcgill.ca.
authors.forename: Jiannis
authors.initials: J
authors.lastname: Ragoussis
authors.affiliation: European Molecular Biology Laboratory (EMBL), Genomics Core Facility, Heidelberg, Germany. benes@embl.de.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Benes
authors.affiliation: McGill Group for Suicide Studies (MGSS), Douglas Mental Health University Institute, McGill University, Frank B Common Pavilion, Room F-2101.2, 6875 LaSalle Boulevard, Montreal, QC, H4H 1R3, Canada. gustavo.turecki@mcgill.ca.|Department of Human Genetics, McGill University, Montreal, QC, Canada. gustavo.turecki@mcgill.ca.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Turecki
authors.affiliation: McGill Group for Suicide Studies (MGSS), Douglas Mental Health University Institute, McGill University, Frank B Common Pavilion, Room F-2101.2, 6875 LaSalle Boulevard, Montreal, QC, H4H 1R3, Canada. carl.ernst@mcgill.ca.|Department of Human Genetics, McGill University, Montreal, QC, Canada. carl.ernst@mcgill.ca.
authors.forename: Carl
authors.initials: C
authors.lastname: Ernst
mesh_terms: D019295:Computational Biology; D005819:Genetic Markers / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D009928:Organ Specificity; D011786:Quality Control; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome
chemical_list: D005819:Genetic Markers; D035683:MicroRNAs