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2015 ; 8
(ä): 35
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Biomarker discovery: quantification of microRNAs and other small non-coding RNAs
using next generation sequencing
#MMPMID26130076
Lopez JP
; Diallo A
; Cruceanu C
; Fiori LM
; Laboissiere S
; Guillet I
; Fontaine J
; Ragoussis J
; Benes V
; Turecki G
; Ernst C
BMC Med Genomics
2015[Jul]; 8
(ä): 35
PMID26130076
show ga
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.