Gut microbiome in hemodialysis patients treated with calcium acetate or treated with sucroferric oxyhydroxide: a pilot study

Purpose It has been proved that the gut microbiome is altered in patients with chronic kidney disease. This contributes to chronic inflammation and increases cardiovascular risk and mortality, especially in those undergoing hemodialysis. Phosphate binders may potentially induce changes in their microbiome. This trial aimed to compare the changes in the gut microbiome of hemodialysis patients treated with calcium acetate to those treated with sucroferric oxyhydroxide. Methods Twelve hemodialysis patients were distributed to receive calcium acetate or sucroferric oxyhydroxide for 5 months. Blood samples (for biochemical analysis) and stool samples (for microbiome analysis) were collected at baseline, 4, 12, and 20 weeks after treatment initiation. Fecal DNA was extracted and a 16S rRNA sequencing library was constructed targeting the V3 and V4 hypervariable regions. Results Regarding clinical variables and laboratory parameters, no statistically significant differences were observed between calcium acetate or sucroferric oxyhydroxide groups. When analyzing stool samples, we found that all patients were different (p = 0.001) among themselves and these differences were kept along the 20 weeks of treatment. The clustering analysis in microbial profiles grouped the samples of the same patient independently of the treatment followed and the stage of the treatment. Conclusion These results suggest that a 5-month treatment with either calcium acetate or sucroferric oxyhydroxide did not modify baseline diversity or baseline bacterial composition in hemodialysis patients, also about the high-variability profiles of the gut microbiome found among these patients.

Reply to Grigoriev et al., “Sequences of SARS-CoV-2 “Hybrids” with the Human Genome: Signs 1 of Non-coding RNA?”

High throughput sequencing reads from virally infected cells provide detailed information about both the infected host cells and invading viruses (1). For example, RNA-sequencing techniques from infected cells contains reads that unequivocally align to either the host or the viral transcriptomes, enabling quantification of host and viral gene expressions (2). Occasionally, there are reads with split characteristics, having one part (e.g., the 5’ end) unambiguously matching the host and another part (e.g., the 3’ end) clearly matching the viral genomes. The split characteristic with unambiguous matching on either part is the key here, typically requiring convincing stretches of sequence matches such as >30bp that we used in our analysis (3). Such reads are termed host-virus chimeric reads (HVCRs). Indeed, HVCRs that surpass statistical reproducibility and signal-to-noise standards might carry novel insights into the biology of host-virus interactions (4, 5). Thus, it is important to unambiguously detect statistically rigorous and biologically relevant HVCRs. We and others have shown that detection of relevant HVCRs is complicated by unfaithful reverse-transcriptase and polymerase enzymes that template-switch during typical high throughput sequencing library preparation protocols (6–9).

First report of Apple rubodvirus 2 infecting pear (Pyrus communis) in South Africa

Apple rubbery wood virus 2 (ARWV-2; Rott et al., 2018) belong to the species Apple rubodvirus 2, a member of the genus Rubodvirus (family Phenuiviridae; Kuhn et al 2020). ARWV-2 was first identified in apples and is associated with apple rubbery wood disease (ARWD) that is characterized by unusual flexibility of stems and branches, reduced growth, shortened internodes and increased cold sensitivity (Jakovljevic et al., 2017, Rott et al., 2018). ARWD was first reported in 1935 in England on apple and has since been found on quince and pear (Jakovljevic et al., 2017; Rott et al., 2018). In January 2021, leaves were collected from a pear tree (Pyrus communis cv. Forelle, F514) in a commercial orchard near Villiersdorp, South Africa. The tree displayed no foliar or tree branch symptoms, except for malformed fruits potentially due to insect feeding damage or pear stony pit disease previously associated with infection of apple stem pitting virus (ASPV) (Paunovic et al. 1999). Leaf petioles (one gram) were used for total RNA extraction, using a modified CTAB extraction protocol (Ruiz-García et al. 2019). A sequencing library was constructed (Illumina TruSeq Stranded Total RNA with plant Ribo-Zero) and sequenced on an Illumina HiseqX instrument (Macrogen, South Korea). A total of 30,709,182 paired-end reads (100 nt) were obtained and trimmed for quality with Trimmomatic (SLIDINGWINDOW:3:20, MINLEN:20) (Bolger et al. 2014). De novo assembly, using default parameters of CLC Genomics Workbench 11.0.1 (Qiagen), resulted in 97,294 contigs. BLASTn analysis identified 17 viral contigs, with 14 contigs having high nucleotide identity to ASPV and three to ARWV-2. The latter contigs included all three segments of ARWV-2. The L contig was 7371 nts, M was 1289 nts and S was 1463 nts in length, generated with 7341, 626 and 9161 reads for segment L, M and S, respectively. Segment S had the highest read coverage (524.87x), followed by segment L (88.07x) and M (36.60x). The ARWV-2 GenBank accessions with the highest percentage identity to the contigs were MF062128.1 from United States of America (98.2% to segment L), MN163134.1 from China (97.5% to segment M) and NC_055535.1 from Germany (93.5% to segment S). The contigs spanned 100%, 80.92% and 100% of these accessions of segments L, M and S, respectively and were deposited in GenBank as accessions MZ593725- MZ593727. Reverse transcription polymerase chain reaction (RT-PCR) was used to validate the presence of ARWV-2 in sample F514, using primers directed at segments L (con708_178F/con708_666R), M (ARWaV-2S1_38F/ARWaV-2S1_682R) and S (ARWaV-2M567F/ARWaV-2M1342R) (Rott et al., 2018). Amplicon sequences (510 bp (L), 645 bp (M) and 799 bp (S)) were confirmed with bi-directional Sanger sequencing. Fifty-nine additional pear samples were surveyed in 2021 for ARWV-2 using the M segment assay as mentioned above. The survey included the Koue Bokkeveld and Elgin areas, and cultivars Bosc (22 samples), Abate (10 samples), Rosemarie (3 samples), Forelle (9 samples), Packham’s Triumph (12 samples) and Early Bon Chretien (3 samples). A total of 27 samples (11 samples from the Koue Bokkeveld region and 16 samples from the Elgin region) tested positive for ARWV-2, demonstrating the common presence of this virus in pears in South Africa. This is the first report of ARWV-2 infecting pear in South Africa. Although no association with disease symptoms were observed, this study expands the data on the incidence and distribution of this virus in South Africa.

Tagmentation-based single-cell genomics

It has been just over 10 years since the initial description of transposase-based methods to prepare high-throughput sequencing libraries, or “tagmentation,” in which a hyperactive transposase is used to simultaneously fragment target DNA and append universal adapter sequences. Tagmentation effectively replaced a series of processing steps in traditional workflows with one single reaction. It is the simplicity, coupled with the high efficiency of tagmentation, that has made it a favored means of sequencing library construction and fueled a diverse range of adaptations to assay a variety of molecular properties. In recent years, this has been centered in the single-cell space with a catalog of tagmentation-based assays that have been developed, covering a substantial swath of the regulatory landscape. To date, there have been a number of excellent reviews on single-cell technologies structured around the molecular properties that can be profiled. This review is instead framed around the central components and properties of tagmentation and how they have enabled the development of innovative molecular tools to probe the regulatory landscape of single cells. Furthermore, the granular specifics on cell throughput or richness of data provided by the extensive list of individual technologies are not discussed. Such metrics are rapidly changing and highly sample specific and are better left to studies that directly compare technologies for assays against one another in a rigorously controlled framework. The hope for this review is that, in laying out the diversity of molecular techniques at each stage of these assay platforms, new ideas may arise for others to pursue that will further advance the field of single-cell genomics.

Mitochondrial mRNA is a stable and convenient reference for the normalization of RNA-seq data

The normalization of high-throughput RNA sequencing (RNA-seq) data is needed to accurately analyze gene expression levels. Traditional normalization methods can either correct the differences in sequencing depth, or correct both the sequencing depth and other unwanted variations introduced during sequencing library preparation through exogenous spike-ins1-4. However, the exogenous spike-ins are prone to variation5,6. Therefore, a better normalization approach with a more appropriate reference is an ongoing demand. In this study, we demonstrated that mitochondrial mRNA (mRNA encoded by mitochondria genome) can serve as a steady endogenous reference for RNA-seq data analysis, and performs better than exogenous spike-ins. We also found that using mitochondrial mRNA as a reference can reduce batch effects for RNA-seq data. These results provide a simple and practical normalization strategy for RNA-seq data, which will serve as a valuable tool widely applicable to transcriptomic studies.

Nuclear isolation and sequencing for mouse hypothalamus v1

This protocol is intended for isolation of nuclei from fresh brain tissue in preparation for single-nuclei sequencing library using Chromium Single Cell 3’ Reagent Kits v2 or v3 (10X Genomics). For each step, tissue and reagents were kept on ice or at 4C, to reduce unwanted gene expression due to procedures and maintain nuclear integrity.

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.

Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.