A method for simultaneous detection of small and long RNA biotypes by ribodepleted 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 extremely efficient, resulting in less than 1.5% rRNA content in the final library. We identified > 30,000 unique transcripts from all samples, including protein-coding genes and many species of non-coding RNA, in biologically-relevant proportions. Furthermore, the normalized sequencing read count for select genes significantly negatively correlated with Ct values from qRT-PCR 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.

hNOP2/NSUN1 Regulates Ribosome Biogenesis through Stabilization of snoRNP Complexes and Cytosine-5 Methylation of 28S rRNA.

5-Methylcytosine (m5C) is a base modification broadly found on a variety of RNAs in the human transcriptome. In eukaryotes m5C is catalyzed by enzymes of the NSUN family, which is composed of seven members in humans (NSUN1-7). NOP2/NSUN1 has been mostly characterized in budding yeast as an essential ribosome biogenesis factor required for the deposition of m5C on the 25S rRNA. Although human NOP2/NSUN1 has been known to be an oncogene overexpressed in several types of cancer, its functions remain poorly characterized. To define the roles of human NOP2/NSUN1, we used an miCLIP-seq approach to identify its RNA substrates. Our analysis reveals that vault RNA 1.2 and rRNA are NOP2/NSUN1-specific methylated targets and we further confirm by bisulfite sequencing that NOP2/NSUN1 is responsible for the deposition of m5C at residue 4447 on the 28S rRNA. Depletion of NOP2/NSUN1 impairs cell proliferation, rRNA processing and 60S ribosome biogenesis. Additionally, we find that NOP2/NSUN1 binds to the 5′ETS region of the pre-rRNA transcript and regulates pre-rRNA processing in part through non-catalytic complex formation with box C/D snoRNAs. Our study identifies for the first time the RNA substrates of human NOP2/NSUN1 and reveals additional functions in rRNA processing beyond catalyzing m5C base modification.

993. A CRISPR-Powered Universal Infectious Disease Assay

Background The COVID-19 pandemic has brought awareness to the dangers of emerging pathogens to global human health and welfare. Sensitivity and flexibility are important features for methods used to detect emerging pathogens. While PCR testing is rapid and sensitive, a significant advantage next generation sequencing (NGS) approaches have over PCR-based analyses is the ability to detect previously undiscovered pathogens while also providing genomic information that can detect SARS-CoV-2 genome sequence, identify source of co-infection, and the host transcriptional response in a single workflow. The critical component enabling this approach is Jumpcode CRISPRclean technology which removes abundant human and bacterial ribosomal RNA sequences from NGS libraries. CRISPRclean workflow easily integrates into next generation sequencing projects Schematic of the Jumpcode CRISPRclean protocol Methods CRISPRclean was applied to contrived infected tissue samples including human lung RNA spiked with serially diluted amounts of SARS-CoV-2 RNA and bacterial RNA. NEB RNA libraries were prepared and treated with CRISPRclean protocol, then sequenced on Illumina instruments. Data analysis was performed using Jumpcode proprietary software to measure alignment and depletion rates, the Silva database for rRNA read alignment, and Kraken2 and CosmosID pipelines for k-mer based metagenomic investigation. Fold enrichment of SARS-CoV-2 reads after CRISPRclean depletion of libraries prepared from contrived samples. CRISPRclean treatment of the fully contrived samples increases the fraction of reads that map to the SARS-CoV-2 genome by an average of ~10-fold Results CRISPRclean treatment of the contrived samples increases ~10 fold of reads that map to the SARS-CoV-2 genome. For the 60 viral copies of SARS-CoV-2 sample, the number of reads mapping to the SARSCoV-2 genome increases from ~10,000 reads to ~70,000 reads. A similar increase in reads occurs for S. aureus. The percentage of SARS-CoV-2 genome covered at 1X and 10X also increases. Similar results were achieved even after downsampling the datasets to 5M reads. There is a ~4-fold increase in bacterial species detection in these stool samples after CRISPRclean treatment. Percentage of SARS-CoV-2 genome covered at 1X and 10X increases as a result of rRNA depletion. Coverage of the SARS-CoV-2 genome at 50 million reads. Number of reads aligning to the S. aureus and SARS-CoV-2 genomes increases after CRISPRclean depletion. For the sample containing 0.0001% SARS-CoV-2, (60 viral copies), the number of reads mapping to the SARS-CoV-2 genome increases from ~10,000 reads to ~70,000 reads. CosmosID Shotgun Metagenomics Analysis heat map showing read alignments to viral genomes. The yellow color indicates high read counts. The CosmosID shotgun metagenomic analysis software was used to analyze the sequencing data, classify the sequences and generate the heat map. Conclusion Metatranscriptomics powered by CRISPR-mediated rRNA depletion offers a robust methodology to acquire viral genomic data, microbiome composition, co-infection information, and the transcriptional status of the host immune response in a single workflow. This sequencing-based approach can be available on the first day of the next viral outbreak and should be considered as a first-line test for novel zoonotic virus detection. Bacterial species composition of patient stool samples before and after CRISPRclean depletion. ~4-fold increase in bacterial species detection in these stool samples after CRISPRclean treatment. Sequencing data downsampled to 20 million reads. Disclosures Keith Brown, n/a, Jumpcode Genomics (Board Member, Employee, Shareholder)

RiboRid: A low cost, advanced, and ultra-efficient method to remove ribosomal RNA for bacterial transcriptomics

RNA sequencing techniques have enabled the systematic elucidation of gene expression (RNA-Seq), transcription start sites (differential RNA-Seq), transcript 3′ ends (Term-Seq), and post-transcriptional processes (ribosome profiling). The main challenge of transcriptomic studies is to remove ribosomal RNAs (rRNAs), which comprise more than 90% of the total RNA in a cell. Here, we report a low-cost and robust bacterial rRNA depletion method, RiboRid, based on the enzymatic degradation of rRNA by thermostable RNase H. This method implemented experimental considerations to minimize nonspecific degradation of mRNA and is capable of depleting pre-rRNAs that often comprise a large portion of RNA, even after rRNA depletion. We demonstrated the highly efficient removal of rRNA up to a removal efficiency of 99.99% for various transcriptome studies, including RNA-Seq, Term-Seq, and ribosome profiling, with a cost of approximately $10 per sample. This method is expected to be a robust method for large-scale high-throughput bacterial transcriptomic studies.

Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries

RNA sequencing (RNA-Seq) experiments focused on gene expression involve removal of ribosomal RNA (rRNA) because it is the major RNA constituent of cells. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing must be performed to compensate for the sequencing reads wasted on rRNA. The ideal RNA enrichment method removes all rRNA without affecting other RNA in the sample. We tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We find that the RNase H depletion method is more efficient in depleting rRNA and more specific in recapitulating non-rRNA levels present in unenriched controls than the commonly-used Poly(A) isolation method. The RNase H depletion method is also more effective than the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding RNA levels present in unenriched controls, while the Ribo-Zero depletion method more closely recapitulates annotated non-coding RNA (ncRNA) levels. Finally, we leverage these data to accurately map the C. neoformans mitochondrial rRNA genes, and also demonstrate that RNA-Seq data generated with the RNase H and Ribo-Zero depletion methods can be used to explore novel C. neoformans long non-coding RNA genes.

Ribocutter: Cas9-mediated rRNA depletion from multiplexed riboseq libraries

RNA sequencing libraries produced from fragmented RNA, especially Ribo-seq libraries, contain high proportions of reads from abundant non-coding RNAs. Here, we describe a streamlined Cas9-based protocol for removing abundant rRNA/ncRNA contaminants from Ribo-seq (or other small RNA-seq) libraries and an easy-to-use software tool, ribocutter, for designing sgRNA templates. Following sgRNA template design, the pool of templates is in vitro transcribed using a 1-step commercial kit, which produces enough sgRNAs for multiple treatments. A single multiplexed sequencing library is then treated with Cas9/sgRNAs, followed by a short PCR program, which can increase the fraction of useful reads by more than 3-fold. Comparison of samples before and after depletion demonstrates that Cas9 produces minimal off-target effects and preserves key features (eg. footprint length, periodicity) of Ribo-seq libraries. The method is thus highly effective, costs <€1.00 per sample, and minimises non-specific depletion and technical variation between samples.

TASPERT: Target-Specific Reverse Transcript Pools to Improve HTS Plant Virus Diagnostics

High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique’s success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept.

A head-to-head comparison of ribodepletion and polyA selection approaches for C. elegans low input RNA-sequencing libraries

A recent and powerful technique is to obtain transcriptomes from rare cell populations, such as single neurons in C. elegans, by enriching dissociated cells using fluorescent sorting. However, these cell samples often have low yields of RNA that present challenges in library preparation. This can lead to PCR duplicates, noisy gene expression for lowly expressed genes, and other issues that limit endpoint analysis. Further, some common resources, such as sequence specific kits for removing ribosomal RNA, are not optimized for non-mammalian samples. To advance library construction for such challenging samples, we compared two approaches for building RNAseq libraries from less than 10 nanograms of C. elegans RNA: SMARTSeq V4 (Takara), a widely used kit for selecting poly-adenylated transcripts; and SoLo Ovation (Tecan Genomics), a newly developed ribodepletion-based approach. For ribodepletion, we used a custom kit of 200 probes designed to match C. elegans rRNA gene sequences. We found that SoLo Ovation, in combination with our custom C. elegans probe set for rRNA depletion, detects an expanded set of noncoding RNAs, shows reduced noise in lowly expressed genes, and more accurately counts expression of long genes. The approach described here should be broadly useful for similar efforts to analyze transcriptomics when RNA is limiting.