Biology subjects

Stanton, J.-A. L.

Publications and source records attributed to Stanton, J.-A. L..

3 recordsLinked to original sources

bioRxiv2024

CRISPR-based environmental biosurveillance assisted via artificial intelligence design of guide-RNAs

Environmental biosecurity challenges are worsening for aquatic ecosystems as climate change and increased anthropogenic pressures facilitate the spread of invasive species, thereby broadly impacting ecosystem composition, functioning, and services. Environmental DNA (eDNA) has transformed traditional biomonitoring through detection of trace DNA fragments left by organisms in their surroundings, primarily by application of the quantitative polymerase chain reaction (qPCR). However, qPCR presents challenges, including limited portability, reliance on precise thermal cycling, and susceptibility to inhibitors. To address these challenges and enable field-deployable monitoring, isothermal amplification techniques such as Recombinase Polymerase Amplification (RPA) paired with Clustered Regularly Interspaced Short Palindromic Repeats and associated proteins (CRISPR-Cas) have been proposed as alternatives. We report here the development of CORSAIR (CRISPR-based envirOnmental biosuRveillance aSsisted via Artificial Intelligence guide-RNAs), that harnesses the programmability of the CRISPR-Cas technology, RPA and the artificial intelligence (AI)-based tool Activity-informed Design with All-inclusive Patrolling of Targets (ADAPT) to deploy a swift RPA-CRISPR-Cas13a-based method that detects eDNA from two invasive species as proof of concept: Sabella spallanzanii and Undaria pinnatifida. CORSAIR showcased a robust, streamlined method augmented by ADAPT, reaching a high specificity when tested against co-occurring species and a 100% agreement with 12 PCR-benchmarked eDNA samples, reaching a sensitivity of 0.34 copies uL-1 in 1 hour with a cost of 3.5 USD per sample; thus highlighting CORSAIR as a powerful environmental biosurveillance platform for environmental nucleic acid detection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/627849v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@b60942org.highwire.dtl.DTLVardef@119e924org.highwire.dtl.DTLVardef@1956bforg.highwire.dtl.DTLVardef@18e18b9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

molecular biology↗

bioRxiv2024

Unlocking Antarctic molecular time-capsules - recovering historical environmental DNA from museum-preserved sponges

Marine sponges have recently emerged as efficient natural environmental DNA (eDNA) samplers. The ability of sponges to accumulate eDNA provides an exciting opportunity to reconstruct contemporary communities and ecosystems with high temporal and spatial precision. However, the use of historical eDNA (heDNA), trapped within the vast number of specimens stored in scientific collections, opens up the opportunity to begin to reconstruct the communities and ecosystems of the past. Here, using a variety of Antarctic sponge specimens stored in an extensive marine invertebrate collection, we were able to recover information on Antarctic fish biodiversity from specimens up to 20 years old. We successfully recovered 64 fish heDNA signals from 27 sponge specimens. Alpha diversity measures did not differ among preservation methods, but sponges stored frozen had a significantly different fish community composition compared to those stored dry or in ethanol. Our results show that we were consistently and reliably able to extract the heDNA trapped within marine sponge specimens, thereby enabling the reconstruction and investigation of communities and ecosystems of the recent past with a spatial and temporal resolution previously unattainable. Future research into heDNA extraction from other preservation methods, as well as the impact of specimen age and collection method will strengthen and expand the opportunities for this novel resource to access new knowledge on ecological change during the last century.

molecular biology↗

bioRxiv2021

A workflow for simultaneous detection of coding and non-coding transcripts by ribosomal RNA-depleted RNA-Seq.

RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was highly efficient, resulting in less than 1.5% rRNA content in the final library, significantly better than other reported rRNA removal techniques. We identified >30,000 unique transcripts from all samples, including protein-coding genes and many unique species of non-coding RNA, in biologically-relevant proportions. Furthermore, normalized sequencing read count for select genes significantly negatively correlated with Ct values from RT-qPCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.

molecular biology↗