APPLICATION NOTE

Innovative small RNA profiling with unique D-Plex
technology on MGI DNBSEQ™ sequencers
Blum A.1, Fang W.2, Schvartzman S.1, Kroonen J.1, Quoilin C.1, Brocart G.1
Diagenode SA, 2MGI.

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The Diagenode D-Plex small RNA DNBSEQ™ kit is a tool designed for the study of the small non-coding transcriptome. It offers key
advantages such as ultra-low input tolerance, liquid-biopsy compatibility and an easy-to-use protocol with minimal hands-on time. By
applying a ligation-free preparation method, the final D-Plex libraries efficiently capture a vast spectrum of small non-coding RNAs while
minimizing inherent ligation biases. Supported by its capability for high quality sequencing on MGI’s DNBSEQ™ platforms, the D-Plex
small RNA DNBSEQ™ kit is exceedingly suitable for discovery of circulating biomarkers in clinically relevant samples

Introduction
Small non-coding RNAs (sncRNAs) offer great hopeas clinical biomarkers in the prediction and early
detection of disease or in the investigation of responseto treatment. This has been highlighted in the context ofseveral medical conditions such as cancer, liver disease,cardiovascular disease, and central nervous systemdisorders, among many others. There are several
types of sncRNAs, of which microRNAs (miRNAs) are
the best known and the most frequently assessed fortheir potential role as biomarkers [1]. Other sncRNA
species, such as PIWI-interacting RNAs (piRNAs),
small nucleolar RNAs (snoRNAs), small nuclear RNAs
(snRNAs) and tRNA-derived small RNAs represent
new potential classes of biomarkers to be exploredas they have proved to be key components for cellular
regulation in some diseases [2], [3].
Next-generation sequencing (NGS) exhibits anunprecedented opportunity to discover and quantify
diverse kinds of sncRNAs. However, currently available
library preparation methods do not provide optimal
approaches to comprehensively analyze the wholesmall RNA spectrum of a sample of interest. In all
small RNA library production protocols, the RNA
fragment needs to be converted into cDNA, attached
to adaptors on both extremities and enriched via DNAamplification to form a suitable library ready for a
successful sequencing run. A main concern of thegenerally used ligation-based methods represents
the heavy bias caused by an uneven affinity of RNA
ligases towards the different template sequences
and 5’-modifications for the addition of sequencingadapters. These libraries show a clear enrichment

for canonical miRNAs harboring 5’-mono-P and 3’OH, which are precisely the biochemical prerequisites
for the commonly used ligases, leading to anunderrepresentation of the other small RNA species
in the sample of interest [4]. Additionally, ligation-based
methods need large amounts of starting materials(often >10 ng total RNA per sample) and require several
purification steps which are challenging for low input
and clinical-relevant circulating RNA samples.
Here, we aimed to optimize a sequencing library strategy,
which (1) is suitable for ultra-low and circulating RNA
inputs, (2) allows capture of the wide diversity of small
RNAs and (3) is compatible for DNBSEQ™ sequencing
on MGI sequencers. The D-Plex technology utilizes
the innovative capture and amplification by tailingand switching, a ligation-free method for RNA library
preparation [5]. The protocol is described in Figure 1.
As a first step, the RNA fragment is polyadenylated andcaptured by a poly-dT primer which contains part of the3’-adapter sequence and serves as the starting point
for the reverse transcription. By applying a templateswitch reaction, the synthesized cDNA strand is thenelongated by the terminal 5’-adapter sequence andcan be directly used for PCR amplification to enrich
the library as a whole. The final library is then readyto be used for the DNA single-strand circularization
step required for DNBSEQ™ sequencing on MGI
sequencers [6]. Together, our D-Plex technology togenerate complex small RNA libraries from clinicalrelevant RNA samples in combination with MGI’s highly
efficient DNBSEQ™ techniques provides a promising
tool for valuable small RNA profiling.

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Results

Figure 1. D-Plex Small RNA DNBSEQ library preparation workflow.
Single stranded RNAs are polyadenylated at their 3’-end and primed
with a poly-dT oligonucleotide containing the terminal 3’-adapter
sequence. The addition of a template-switching oligonucleotide
enables the elongation of the cDNA synthesis to fuse the terminal
5’-adapter sequence during the reverse transcription reaction.
During PCR amplification, the required MGI’s adapter sequences
and barcodes are incorporated into the final library construct.

With more than 40 million reads per sample, the overall
sequencing quality has exceeded expectations. Even
before trimming of the A-tail, a very high per basesequence quality was achieved with an average scoreof 37 (Figure 2). Still 84%-92% of the reads remained
after trimming and filtering with up to 70% mapping
efficiency (Figure 3). Regarding the capture of the wholesmall non-coding RNA spectrum, detection of a large
diversity of small RNAs could be confirmed (Figure 4).
By efficiently incorporating miRNA, miscRNA, scaRNA,scRNA, snoRNA and snRNA in the libraries, the kit
provides a high potential tool for clinical biomarker
discovery. Importantly, the widespread diversityof information is maintained equally between thereplicates and keeps further consistency even whendecreasing the starting material from 2.5 ng down
to ultra-low 10 pg circulating RNAs derived fromhuman plasma samples. Outstanding ultra-low input
performance could be additionally demonstrated by
calculating the Pearson correlation coefficient between
very low (10 pg, 25 pg and 50 pg) and high (2.5 ng) RNA
inputs, convincing with an outcome of R=0.99 as shownin Figure 5 for the 2.5 ng to 25 pg comparison.

Methods
Circulating RNA was extracted from human plasma(Seralab 23134, lot BRH1462944) using the Qiagen
miRNeasy Mini Kit (Qiagen, 217004). The D-Plex
small RNA DNBSEQ™ (Diagenode, C05030051 (Coremodule – 24 rxns), C05030060 (D-Plex 24 DNBSEQ
Barcodes - Set A), C05030061 (D-Plex 24 DNBSEQ
Barcodes - Set B)) protocol was followed to prepare
RNA libraries and challenged by comparing very-lowinputs (10 pg, 25 pg and 50 pg) with a higher reference
input (2.5 ng). Circularization (MGIEasy Circularization
Kit (MGI, 1000005259)), DNB production and sequencing
were accomplished according to the corresponding
MGI’s protocols (High-throughput Sequencing Set(DNBSEQ-G400RS FCL SE50) (MGI, 1000017992)) ona DNBSEQ-G400 sequencer using the new CoolMPSsequencing technology, SE50 starting with 3 darkcycles. RNA-seq data were processed according
to to the manufacturer’s instructions (Diagenode,C05030060).

Figure 2. Per base sequence quality before trimming.
Data from 50 pg circulating RNAs derived from human plasma
samples.

www.diagenode.com Please contact us for more information
Europe Diagenode sa / LIEGE SCIENCE PARK // Rue du Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // E-mail: info@diagenode.com
USA Diagenode Inc. / 400 Morris Avenue, Suite 101 // Denville, NJ 07834 // USA // Phone: (+1) 862 209-4680 // Mail: info.na@diagenode.com

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APPLICATION NOTE

Figure 3. Sequencing statistics.

Figure 5. Ultra-low input performance.

Filtering and mapping outputs for all RNA inputs (10 pg, 25 pg, 50pg, and 2.5 ng) derived from human plasma samples.

Person correlation between ultra-low (25 pg) and high (2.5 ng)
RNA inputs derived from human plasma samples considering thenormalized counts of small non-coding transcripts.

Conclusion
The D-Plex Small RNA DNBSEQ™ library
preparation protocol represents a fast and easyto-use, one-day one-tube method for unbiased
small non-coding RNA library production withultra-low input capability. Supported by highquality sequencing on MGI’s DNBSEQ™ platforms,
the D-Plex Small RNA DNBSEQ™ kit is suitablefor biomarker discovery in clinically relevantsamples.

References
Figure 4. Diversity of small non-coding RNAs.
Biotype profiles for all RNA inputs (10 pg, 25 pg, 50 pg, and 2.5 ng)
derived from human plasma samples.

[1] Lopez, J. P. et al. (2015) DOI: 10.1186/s12920-015-0109-x[2] Watson, C. N. et al. (2019) DOI: 10.3389/fgene.2019.00364[3] Li, C. H. et al. (2015) DOI: 10.2174/13892029166661507071
55851[4] Meistertzheim, M. et al. (2019) DOI: 10.1373/
clinchem.2019.305045[5] Turchinovich, A. et al. (2014) DOI: 10.4161/rna.29304[6] Drmanac, S. et al. (2020) DOI: 10.1101/2020.02.19.953307

www.diagenode.com Please contact us for more information
Europe Diagenode sa / LIEGE SCIENCE PARK // Rue du Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // E-mail: info@diagenode.com
USA Diagenode Inc. / 400 Morris Avenue, Suite 101 // Denville, NJ 07834 // USA // Phone: (+1) 862 209-4680 // Mail: info.na@diagenode.com

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