Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
[Preprint]. 2024 May 31:2024.05.28.596343.
doi: 10.1101/2024.05.28.596343.

Efficient small fragment sequencing of human, cow, and bison miRNA, small RNA or csRNA-seq libraries using AVITI

Anna L McDonald  1 , Andrew M Boddicker  2 , Marina I Savenkova  1 , Ian M Brabb  1 , Xiaodong Qi  2 , Daniela D Moré  3   4 , Cristina W Cunha  3   4 , Junhua Zhao  2 , Sascha H Duttke  1
Affiliations

Efficient small fragment sequencing of human, cow, and bison miRNA, small RNA or csRNA-seq libraries using AVITI

Anna L McDonald et al. bioRxiv. .

Update in

Abstract

Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina's sequencing by synthesis method has long been essential for NGS but emerging technologies like Element Biosciences' sequencing by avidity (AVITI) represent a novel approach. It has been reported that AVITI offers improved signal-to-noise ratios and cost reductions. However, the method relies on rolling circle amplification which can be impacted by polymer size, raising questions about its efficacy sequencing small RNAs (sRNA) molecules including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and others that are crucial regulators of gene expression and involved in various biological processes. In addition, capturing capped small RNAs (csRNA-seq) has emerged as a powerful method to map active or "nascent" RNA polymerase II transcription initiation in tissues and clinical samples. Here, we report a new protocol for seamlessly sequencing short DNA fragments on the AVITI and demonstrate that AVITI and Illumina sequencing technologies equivalently capture human, cattle (Bos taurus) and the bison (Bison bison) sRNA or csRNA sequencing libraries, augmenting the confidence in both approaches. Additionally, analysis of generated nascent transcription start sites (TSSs) data for cattle and bison revealed inaccuracies in their current genome annotations and highlighted the possibility and need to translate small RNA sequencing methodologies to livestock. Our accelerated and optimized protocol therefore bridges the advantages of AVITI sequencing and critical methods that rely on sequencing short DNA fragments.

Keywords: AVITI; Illumina; capped small RNA sequencing (csRNA-seq); livestock; small RNA sequencing (sRNA-seq).

PubMed Disclaimer

Conflict of interest statement

COMPETING INTERESTS A.M.B, X.Q., and J.Z are employees of Element Biosciences. S.H.D. is leading nascent Transcriptomic Services (nTSS) at Washington State University.

Figures

Fig. 1:
Fig. 1:. Study design.
Small RNAs sizes 18–60 nt were purified from total RNA isolated from human cancer cell lines (A375, BT474), cattle and bison blood. All sRNAs (sRNA-seq) as well as 5’meG cap-enriched RNAs (csRNA-seq) libraries were generated and sequenced on the AVITI and Illumina NGS platforms.
Fig. 2:
Fig. 2:. Uniform sequencing of small and capped small RNA-seq libraries on the Illumina and AVITI platforms.
A. Read length distribution plots of A375 small RNAs sequenced natively on the Illumina and after benchtop circularization (Adapt Rapid PCR-free) on the AVITI platform. The area under each line sums to a total of 100%. Differences between Illumina and AVITI are plotted in grey. B. Read length distribution plots of A375 cells capped small RNAs. C. Scatterplot comparing the expression level of small RNAs and D. capped small RNAs using the Illumina and AVITI platform. E. Comparison of the detection of small RNA types of different lengths (miRNAs: 21–24; snoRNAs: 55–61).
Fig. 3:
Fig. 3:. csRNA-seq facilitates improved annotation of livestock genomes
A. Comparison of experimentally defined TSSs from human A375 cancer cells, B. cattle, and C. bison by csRNA-seq relative to the RefSeq annotation. D. Comparison of the frequency of TATA box sites per 1000 bp between our experimental TSS and RefSeq for human, E. cattle, and F. bison. G. Comparison of the frequency of Initiator sites per 1000 bp between our experimental TSS and RefSeq for human, H. cattle, and I. bison.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Affymetrix CSHL, Encode Transcriptome Project. 2009. Post-transcriptional processing generates a diversity of 5′-modified long and short RNAs. Nature 457: 1028. - PMC - PubMed
    1. Arslan S Garcia FJ Guo M Kellinger MW Kruglyak S LeVieux JA Mah AH Wang H Zhao J Zhou et al. 2024. Sequencing by avidity enables high accuracy with low reagent consumption. Nature Biotechnology 42: 132–138. - PMC - PubMed
    1. Ceasar SA, Rajan V, Prykhozhij SV, Berman JN, Ignacimuthu S. 2016. Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1863: 2333–2344. - PubMed
    1. Chen X. 2009. Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol 25: 21–44. - PMC - PubMed
    1. Dupuis-Sandoval F, Poirier M, Scott MS. 2015. The emerging landscape of small nucleolar RNAs in cell biology. Wiley Interdiscip Rev RNA 6: 381–397. - PMC - PubMed

Publication types