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. 2024 Nov 29;25(1):1157.
doi: 10.1186/s12864-024-11013-7.

Efficient small fragment sequencing of human, cattle, 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  5
Affiliations

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

Anna L McDonald et al. BMC Genomics. .

Abstract

Background: Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina's sequencing by synthesis method has long been central to NGS, but new sequencing methods like Element Biosciences' AVITI technology are emerging. AVITI is reported to offer improved signal-to-noise ratios and cost reductions. However, its reliance on rolling circle amplification, which can be affected by polymer size, raises questions about its effectiveness in sequencing small RNAs (sRNAs) such as microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), and many others. These sRNAs are crucial regulators of gene expression and involved in various biological processes. Additionally, capturing capped small RNAs (csRNA-seq) is a powerful method for mapping active or "nascent" RNA polymerase II transcription initiation in tissues and clinical samples.

Results: Here, we report a new protocol for seamlessly sequencing short fragments on the AVITI and demonstrate that AVITI and Illumina sequencing technologies equivalently capture human, cattle (Bos taurus), and bison (Bison bison) sRNA or csRNA sequencing libraries, increasing confidence in both sequencing approaches. Additionally, analysis of generated nascent transcription start site (TSS) data for cattle and bison revealed inaccuracies in their current genome annotations, underscoring the potential and necessity to translate small and nascent RNA sequencing methodologies to livestock.

Conclusions: Our accelerated and optimized protocol bridges the advantages of AVITI sequencing with critical methods that rely on sequencing short fragments. This advance bolsters the utility of AVITI technology alongside traditional Illumina platforms, offering new opportunities for NGS applications.

Keywords: AVITI; Capped small RNA sequencing (csRNA-seq); Illumina; Livestock; Small RNA sequencing (sRNA-seq).

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Conflict of interest statement

Declarations. Ethical approval and conset to participate: Blood collection from experimental animals followed protocols approved by the Institutional Animal Care and Use Committee of Washington State University (Animal Subject Approval Form #7080 for cattle) or University of Wyoming (Protocol # 20230201BW00287-01 for bison). Consent for publication: Not Applicable. 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 were purified from total RNA isolated from human cancer cell lines (BT474, A375), cattle and bison blood. Libraries containing all small RNAs (sRNA-seq) as well as 5’meG cap-enriched small RNAs that are associated with actively initiating RNA polymerase II (csRNA-seq) of size 20–60 nt were 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 BT474 small RNAs sequenced natively on the Illumina and the AVITI platform using the Cloudbreak Freestyle method. 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 BT474 cells capped small RNAs. C. Scatterplot comparing the expression level (rlog transformed read counts) 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 F)
Fig. 3
Fig. 3
csRNA-seq facilitates improved genome annotations. (A) Comparison of experimentally defined TSSs from human BT474 cancer cells, (B) cattle, and (C) bison by csRNA-seq sequenced using AVITI 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
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