Application Note: RNA Sequencing

Complete Small RNA Sequencing Solutions on theMiniSeq™ System
Uncover the complete picture of small RNA expression regulation with the power of nextgeneration sequencing in a simple, personal platform.
Highlights

Drosha• Efficient, Multiplexed Small RNA Sequencing
Accelerate your research by studying thousands of small
RNA sequences simultaneously with greater sensitivity anddynamic range

pre-miRNAs

Dicer
small RNA

• Accessible, Affordable NGS Technology
Access a personal, reliable sequencing platform for lowthroughput RNA sequencing solutions• Simple, Streamlined End-to-End Solution
Integrated workflows from library preparation to easy pushbutton data analysis options• Industry-Leading Data Quality
Build confidence in your results with the most widely adopted,and proven Illumina sequencing chemistry1

Figure 1: Small RNA Sequencing—Study thousands of miRNA and small RNA
sequences on the MiniSeq System with greater sensitivity and dynamic range.

Introduction
Small RNAs, including microRNAs (miRNAs), are single stranded
noncoding RNAs between 17-34 nucleotides in length and have been
implicated as key gene regulators in many cellular pathways (Figure 1).
Small RNAs are involved in the regulation of numerous cellularprocesses including circadian rhythms and viral regulation.1,2 TheMiniSeq System offers the easiest and most affordable entry into therealm of next-generation sequencing (NGS) for small RNA sequencing(RNA-Seq) applications.

Small RNA Sequencing

NGS makes sequence-based expression analysis a “digital” alternative
to analog techniques. Microarray gene expression measurements arelimited by noise and signal saturation. In contrast, next-generation
sequencing quantifies discrete, digital read counts, offering a large

Prepare LibrarymRNARISC

dynamic range. This enables highly scalable sensitivity, allowing
researchers to tune the level of resolution to meet specificexperimental needs. With the MiniSeq Small RNA-Seq workflow,
millions of small RNA and microRNA (miRNA) sequences can bequeried with unprecedented sensitivity and dynamic range. In addition
to expression and profiling applications, the MiniSeq System is a
powerful tool for the discovery of novel miRNAs and other small
noncoding RNAs. Even without a reference sequence or secondary
structure information, small RNA-Seq allows the characterization ofvariations such as isomiRs (miRNA variants) at single-base resolution.

Simple, Integrated Workflow
The MiniSeq System Small RNA-Seq workflow delivers a complete
sample-to-answer workflow for the analysis of miRNAs and othernoncoding RNAs (Figure 2).

Sequence

Analyze Data

1 day

4 hours2 – 3 hours

TruSeq® Small RNA Library Prep

MiniSeq SystemLocal Run Manager Small RNA Analysis
BaseSpace® Small RNA Apps

Figure 2: MiniSeq Small RNA-Seq Workflow—The integrated workflow enables streamlined library preparation for cost-effective studies covering all small RNA
transcripts in any species.
For Research Use Only. Not for use in diagnostic procedures.

Application Note: RNA Sequencing

Advantages of RNA-Seq Over Microarrays•

Requires no prior knowledge of the transcriptome•

Provides qualitative and quantitative data•

Enables deep sequencing for greater sensitivity and virtuallyunlimited dynamic range•

Delivers sequence and variant information•

Provides greater accuracy of fold-change measurements•

A better value than array technologies, delivering more data
at a lower price per sample

Library Preparation
The small RNA-Seq workflow begins with TruSeq Small RNA LibraryPrep Kit, which provides all reagents required to prepare the RNA
libraries for sequencing, including up to 48 individual indexes formultiplexing. The TruSeq Small RNA Library Preparation Kits provide
a simple, cost- effective method for generating small RNA libraries
directly from total RNA (Figure 3). Small RNAs generated by Dicer
processing are efficiently targeted by modified adapters. The kits also
support customizable size selection to investigate any small RNA
between 17-35 nucleotides. These kits also enable multiplexing withup to 48 unique indexes, allowing efficient study design and maximum
throughput for microRNA and small RNA sequencing runs. Indexes
are added in a universal amplification reaction, greatly reducing ligation
bias and ensuring accurate measurement of small RNA expression.

Figure 3: TruSeq Small RNA-Seq Library Preparation Chemistry—TheTruSeq Small RNA Library Preparation Kits provide a simple, cost- effective
method for generating small RNA libraries directly from total RNA.

Sequencing on the MiniSeq System
The MiniSeq System is designed for easy, streamlined operation(Figure 4). For quick, load-and-go set up, the MiniSeq Reagent Kits
provide a flow cell, wash reagents, and a single reagent cartridge
preloaded with all required sequencing reagents. The MiniSeq Reagent
Kits are available in Mid-Output and High-Output formats, allowing
optimization of study designs based on read-length, sample number,and output requirements.
The MiniSeq System offers an intuitive touch screen interface
that provides simple, step-by-step guidance through each stageof the sequencing run, including library and reagent loading, runconfiguration, and run monitoring. MiniSeq Control Software performs
onboard image analysis, base calling, and quality scoring. Quality
statistics from 1 or multiple runs can be monitored in real time using
the Sequencing Analysis Viewer (SAV) software. SAV software can beused onboard the sequencing instrument, or can be accessed fromany location with a Windows-based PC.

Simplified Bioinformatics
Data analysis with the MiniSeq System requires no informatics
expertise or command-line experience. The MiniSeq System features
Local Run Manager software, an on-instrument system for creating arun, monitoring status, and analyzing sequencing data. With Local Run
Manager, on-instrument data analysis can be automatically performed
upon completion of the sequencing run. The data analysis modules
generate simple reports for a wide range of sequencing applications.

For Research Use Only. Not for use in diagnostic procedures.

Figure 4: MiniSeq System—The MiniSeq System leverages the latest advancesin Illumina sequencing chemistry and an easy, integrated workflow.

Table 1: Small RNA-Seq Sample Throughput on the MiniSeq SystemStudy
Measurement

Reads PerSample

Differential expression of allsmall RNAs in a sample

2M(1 × 50 bp)

Samples Per Run
8M25 M412

Application Note: RNA Sequencing

Figure 6: Small RNA Length Distribution—Review small RNA length
distribution with the Small RNA v1.0 App in BaseSpace

Figure 5: Small RNA-Seq Analysis in BaseSpace—The Small RNA v1.0 App
in BaseSpace enables visualization of small RNA precursors, mature miRNAs,and isomiR hits.

The modular design allows users to install and update individual
analysis modules as needed. In addition, sequencing data generated
with the MiniSeq System can be instantly transferred, stored, andanalyzed in the BaseSpace Computing Environment (cloud-based oronsite). BaseSpace RNA-Seq software Apps provide expert-preferreddata analysis tools packaged in an intuitive, click-and-go user interface
designed for informatics novices. The Small RNA BaseSpace App3aligns reads and performs optional novel precursor discovery as wellas pairwise differential expression analysis (Figure 5 and Figure 6).
Pairwise differential expression analysis identifies differentiallyexpressed miRNAs, precursor groups, miRNA families, and piRNAs foreach pair of sample groups.
For downstream analysis, BaseSpace Apps generate output files thatcan be directly input into a broad range of data analysis tools. TheBaseSpace Environment includes a growing community of developers
who use and provide software tools for visualization, analysis, andsharing. This NGS ecosystem provides one of the largest collections ofcommercial and open-source analysis tools currently available.

Small RNA-Seq vs Microarrays
Compared to microarrays, RT-PCR, and other traditional technologies,
NGS small RNA-Seq offers several advantages. Small RNA-Seq allows
deep sequencing, which provides higher sensitivity and a virtuallyunlimited dynamic range. This sensitivity enables superior performanceand greater accuracy of fold-change measurements. Furthermore,sequencing provides in-depth, base-by-base coverage over the areaof interest, which offers a more comprehensive approach for discovery
applications or for the measurement of rare transcripts compared to
traditional methods.

Summary
The MiniSeq System Small RNA-Seq Solution combines innovativeengineering with proven sequencing chemistry to deliver a clear andcomplete view of miRNAs and other small RNAs. With the smallest
footprint of any Illumina sequencing platform, the MiniSeq makes
NGS technology more accessible to more researches and laboratories
than ever before. By harnessing the power of NGS for small RNA
sequencing, researchers can improve the accuracy and sensitivityof their data while simultaneously reducing costs. Accelerate yourresearch and obtain your goals sooner with the MiniSeq Small RNA
Sequencing Solution.

Learn More
To learn more about small RNA-Seq, go to
www.illumina.com/techniques/sequencing/rna-sequencing/small-rnaseq.html.
For more on the TruSeq Small RNA Library Prep Kit, go to
www.illumina.com/products/truseq_small_rna_sample_prep_kit.html.

Ordering Information
Sequencing System

Catalog No.

MiniSeq System

SY-420-1001

Sequencing Kits
MiniSeq High Output Kit (75 Cycles)

FC-420-1001

MiniSeq Mid Output Kit (300 Cycles)

FC-420-1004

Small RNA Library Prep Kitsa
TruSeq Small RNA Library Prep Kit – Set A (24 samples)

RS-200-0012

TruSeq Small RNA Library Prep Kit – Set B (24 samples)

RS-200-0024

TruSeq Small RNA Library Prep Kit – Set C (24 samples)

RS-200-0036

TruSeq Small RNA Library Prep Kit – Set D (24 samples)

RS-200-0048

a. 24 sample kits contain 12 indexes

For Research Use Only. Not for use in diagnostic procedures.

Application Note: RNA Sequencing

References
1.

2.

3.

Flintoft L. MicroRNAs needed for time delay. Nature Reviews Genetics.
2013;14:746 (www.nature.com/nrg/journal/v14/n11/full/nrg3611.html).
Accessed15 December 2015.
Lusic M, Marini B, Ali H, Lucic B, Luzzati R, Giacca M. Proximity to PML
nuclear bodies regulates HIV-1 latency in CD4+ T cells.
Cell Host Microbe. 2013;13:665-7.
Small RNA BaseSpace App (www.illumina.com/informatics/research/sequencing-data-analysis-management/basespace/basespace-apps/smallrna-948948.html). Accessed on 15 December 2015.

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For Research Use Only. Not for use in diagnostic procedures.
© 2016 Illumina, Inc. All rights reserved. Illumina, BaseSpace, MiniSeq, TruSeq, and the pumpkin orange color are trademarks of Illumina,
Inc. and/or its affiliate(s) in the U.S. and/or other countries. Pub. No. 770-2015-045 Current as of 08 January 2016