Small RNA (sRNA) sequencing has been critical for our understanding of many cellular processes, including gene regulation. Nonetheless, the varying biochemical properties of sRNA, such as 5´ nucleotide modifications, make many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy that captures a more complete picture of sRNA. By tagging 5´P sRNA during library preparation, 5XP-seq combines an open approach that includes all types of 5ʹ-terminal modifications (5´X), with a selective approach for 5-phosphorylated sRNA (5´P). We show that 5XP-seq not only enriches phosphorylated miRNA and piRNA but successfully discriminates these sRNA from all other sRNA species. We further demonstrate the importance of this strategy by successful inter-species validation of sRNAs that would have otherwise failed, including human to insect translation of several tRNA (tRFs) and rRNA (rRFs) fragments. By combining 5´ insensitive library strategies with 5´ sensitive tagging, we have successfully tackled an intrinsic bias in modern sRNA sequencing that will help us reveal the true complexity and the evolutionary significance of the sRNA world.

Small RNA (sRNA) sequencing has been critical for our understanding of the transcriptional regulation in most biological processes. Nonetheless, the varying biochemical properties of sRNA, such as 5' nucleotide modifications, make many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy on how to overcome this limitation. By specifically tagging 5'-phosphorylated sRNA (5'P sRNA), that enriches miRNA and piRNA, we successfully discriminate these sRNA from all other sRNA species. Our results underscore the importance of more holistic approaches, in which 5' insensitive library strategies are combined with 5' sensitive tagging. Further advances using such strategies will overcome the increasing bias in reports of easily caught sRNA and ensure assessment of the previously overlooked transcriptome.

Small RNA sequencing (sRNA-seq) has become important for studying regulatory mechanisms in many cellular processes. Data analysis remains challenging, mainly because each class of sRNA--such as miRNA, piRNA, tRNA- and rRNA- derived fragments (tRFs/rRFs)--needs special considerations. Analysis therefore involves complex workflows across multiple programming languages, which can produce research bottlenecks and transparency issues. To make analysis of sRNA more accessible and transparent we present seqpac: a tool for advanced group-based analysis of sRNA completely integrated in R. This opens advanced sRNA analysis for Windows users--from adaptor trimming to visualization. Seqpac provides a framework of functions for analyzing a PAC object, which contains 3 standardized tables: sample phenotypic information (P), sequence annotations (A), and a counts table with unique sequences across the experiment (C). By applying a sequence-based counting strategy that maintains the integrity of the fastq sequence, seqpac increases flexibility and transparency compared to other workflows. It also contains an innovative targeting system allowing sequence counts to be summarized and visualized across sample groups and sequence classifications. Reanalyzing published data, we show that seqpac's fastq trimming performs equal to standard software outside R and demonstrate how sequence-based counting detects previously unreported bias. Applying seqpac to new experimental data, we discovered a novel rRF that was down-regulated by RNA pol I inhibition (anticancer treatment), and up-regulated in previously published data from tumor positive patients. Seqpac is available on github (https://github.com/Danis102/seqpac), runs on multiple platforms (Windows/Linux/Mac), and is provided with a step-by-step vignette on how to analyze sRNA-seq data.

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SUMMARYEarly-life stress can generate persistent life-long effects that impact adult health and disease risk, but little is known of how such programming is established and maintained. Previous use of the Drosophila strain wm4h show that an early embryonic heat shock result in stable epigenetic alteration in the adult fly. To investigate the potential role of small non-coding RNA (sncRNA) in the initiation of such long-term epigenetic effects, we here generated a fine timeline of sncRNA expression during the first 5 stages of Drosophila embryogenesis in this strain. Building on this, we show that (1) miRNA is increased following early embryonic heat shock, and (2) the increased miRNA is coming from two separate sources, maternal and zygotic. By performing long RNA sequencing on the same single embryo, we found that a subgroup of miRNA with maternal origin, had a strong negative correlation with a group of early zygotic transcripts. Critically, we found evidence that one such early zygotic transcript, the insulator binding factor Elba1, is a Su(var) for wm4h. The findings provide insights of the dynamics and stress-sensitivity of sncRNA during the first embryonic stages in Drosophila and suggest an interplay between miRNA, Elba1 and long-term epigenetic alteration.HIGHLIGHTSWe provide a high-resolution timeline for sncRNA for Drosophila stage 1-5 embryosHeat shock before midblastula transition (MBT) results in a massive upregulation of miRNA at cellularizationHeat shock-induced miRNAs negatively associate with downregulation of a specific subset of pre-MBT genesElba1 is a position-effect-variegation (PEV) modifier for wm4hHeat shock-induces an “leaky” expression of genes that overlap with Elba 1-3 binding sites

A wide spectrum of exogenous factors, including diet, environmental pollutants, stress, and seasonal changes have major impact on sperm quality and function. The molecular basis, however, that explains this susceptibility remains largely unknown. Using a combination of proteomics and small RNA (sRNA) sequencing, we show that Drosophila sperm display rapid molecular changes in response to dietary sugar, both in terms of metabolic/redox proteins and sRNA content, particularly miRNA and mitochondria derived sRNA (mt-sRNA). Thus, results from two independent omics point at the dynamics of mitochondria as the central aspect in rapid metabolic adjustments in sperm. Using specific stains and in vivo redox reporter flies, we show that diet indeed rapidly alters the production of mitochondrial derived reactive oxygen species (ROS). Quenching ROS via supplementation of N acetyl cysteine reduces diet-upregulated miRNA, but not mitochondrial-sRNA. Together, these results open new territories in our search for the mechanistic understanding of sperm health and disease.

Motivation Feature-based counting is commonly used in RNA-sequencing (RNA-seq) analyses. Here, sequences must align to target features (like genes or non-coding RNAs) and related sequences with different compositions are counted into the same feature. Consequently, sequence integrity is lost, making results less traceable against raw data. Implementation Small RNA (sRNA) often maps to multiple features and shows an incredible diversity in form and function. Therefore, applying feature-based strategies may increase the risk of misinterpretation. We present a strategy for sRNA-seq analysis that preserves the integrity of the raw sequence making the data lineage fully traceable. We have consolidated this strategy into Seqpac: An R package that makes a complete sRNA analysis available on multiple platforms. Using published biological data, we show that Seqpac reveals hidden bias and adds new insights to studies that were previously analyzed using feature-based counting. Conclusions We have identified limitations in the concurrent analysis of RNA-seq data. We call it the traceability dilemma in alignment-based sequencing strategies. By building a flexible framework that preserves the integrity of the read sequence throughout the analysis, we demonstrate better interpretability in sRNA-seq experiments, which are particularly vulnerable to this problem. Applying similar strategies to other transcriptomic workflows may aid in resolving the replication crisis experienced by many fields that depends on transcriptome analyses. Availability Seqpac is available on Bioconductor (https://bioconductor.org/packages/seqpac) and GitHub (https://github.com/danis102/seqpac). Supplementary information Supplementary data are available at Bioinformatics online.

Early‐life stress can result in life‐long effects that impact adult health and disease risk, but little is known about how such programming is established and maintained. Here, we show that such epigenetic memories can be initiated in the Drosophila embryo before the major wave of zygotic transcription, and higher‐order chromatin structures are established. An early short heat shock results in elevated levels of maternal miRNA and reduced levels of a subgroup of zygotic genes in stage 5 embryos. Using a Dicer‐1 mutant, we show that the stress‐induced decrease in one of these genes, the insulator‐binding factor Elba1, is dependent on functional miRNA biogenesis. Reduction in Elba1 correlates with the upregulation of early developmental genes and promotes a sustained weakening of heterochromatin in the adult fly as indicated by an increased expression of the PEV w m4h reporter. We propose that maternal miRNAs, retained in response to an early embryonic heat shock, shape the subsequent de novo heterochromatin establishment that occurs during early development via direct or indirect regulation of some of the earliest expressed genes, including Elba1.