NGI offers spatial transcriptomics through the 10X Genomics Visium method, a technique recently nominated as Method of the Year by Nature Methods. The technology combines histology with unbiased transcriptomics in a spatial context.
The Illumina NovaSeq 6000 system is the largest of the Illumina sequencing instruments, able to run two flow cells independently of each other and generate massive sequencing depth at competitive prices.
NGI is still up and running during the Covid-19 pandemic, but we are experiencing some limitations in terms of personnel and key reagents. Each NGI node is following its respective host university recommendations and will continue operation until further notice.
Cytogenetically visible inversions are formed by multiple molecular mechanisms.
M Pettersson, CM Grochowski, J Wincent, J Eisfeldt, AM Breman, SW Cheung, ACV Krepischi, C Rosenberg, JR Lupski, J Ottosson, L Lovmar, J Gacic, ES Lundberg, D Nilsson, CMB Carvalho, A Lindstrand
Hum. Mutat., 41 (11) 1098-1004 (2020)
Cytogenetically detected inversions are generally assumed to be copy number and phenotypically neutral events. While nonallelic homologous recombination is thought to play a major role, recent data suggest the involvement of other molecular mechanisms in inversion formation. Using a combination of short-read whole-genome sequencing (WGS), 10X Genomics Chromium WGS, droplet digital polymerase chain reaction and array comparative genomic hybridization we investigated the genomic structure of 18 large unique cytogenetically detected chromosomal inversions and achieved nucleotide resolution of at least one chromosomal inversion junction for 13/18 (72%). Surprisingly, we observed that seemingly copy number neutral inversions can be accompanied by a copy-number gain of up to 350 kb and local genomic complexities (3/18, 17%). In the resolved inversions, the mutational signatures are consistent with nonhomologous end-joining (8/13, 62%) or microhomology-mediated break-induced replication (5/13, 38%). Our study indicates that short-read 30x coverage WGS can detect a substantial fraction of chromosomal inversions. Moreover, replication-based mechanisms are responsible for approximately 38% of those events leading to a significant proportion of inversions that are actually accompanied by additional copy-number variation potentially contributing to the overall phenotypic presentation of those patients.
An efficient single-cell transcriptomics workflow for microbial eukaryotes benchmarked on Giardia intestinalis cells
H Onsbring, AK Tice, BT Barton, MW Brown, TJG Ettema
BMC Genomics, 21 (1) 1471-2164 (2020)
Most diversity in the eukaryotic tree of life is represented by microbial eukaryotes, which is a polyphyletic group also referred to as protists. Among the protists, currently sequenced genomes and transcriptomes give a biased view of the actual diversity. This biased view is partly caused by the scientific community, which has prioritized certain microbes of biomedical and agricultural importance. Additionally, some protists remain difficult to maintain in cultures, which further influences what has been studied. It is now possible to bypass the time-consuming process of cultivation and directly analyze the gene content of single protist cells. Single-cell genomics was used in the first experiments where individual protists cells were genomically explored. Unfortunately, single-cell genomics for protists is often associated with low genome recovery and the assembly process can be complicated because of repetitive intergenic regions. Sequencing repetitive sequences can be avoided if single-cell transcriptomics is used, which only targets the part of the genome that is transcribed.
In this study we test different modifications of Smart-seq2, a single-cell RNA sequencing protocol originally developed for mammalian cells, to establish a robust and more cost-efficient workflow for protists. The diplomonad Giardia intestinalis was used in all experiments and the available genome for this species allowed us to benchmark our results. We could observe increased transcript recovery when freeze-thaw cycles were added as an extra step to the Smart-seq2 protocol. Further we reduced the reaction volume and purified the amplified cDNA with alternative beads to test different cost-reducing changes of Smart-seq2. Neither improved the procedure, and reducing the volumes by half led to significantly fewer genes detected. We also added a 5' biotin modification to our primers and reduced the concentration of oligo-dT, to potentially reduce generation of artifacts. Except adding freeze-thaw cycles and reducing the volume, no other modifications lead to a significant change in gene detection. Therefore, we suggest adding freeze-thaw cycles to Smart-seq2 when working with protists and further consider our other modification described to improve cost and time-efficiency.
The presented single-cell RNA sequencing workflow represents an efficient method to explore the diversity and cell biology of individual protist cells.
Labile Dissolved Organic Matter Compound Characteristics Select for Divergence in Marine Bacterial Activity and Transcription.
B Pontiller, S Martínez-García, D Lundin, J Pinhassi
Front Microbiol, 11 1664-302X (2020)
Bacteria play a key role in the planetary carbon cycle partly because they rapidly assimilate labile dissolved organic matter (DOM) in the ocean. However, knowledge of the molecular mechanisms at work when bacterioplankton metabolize distinct components of the DOM pool is still limited. We, therefore, conducted seawater culture enrichment experiments with ecologically relevant DOM, combining both polymer and monomer model compounds for distinct compound classes. This included carbohydrates (polysaccharides vs. monosaccharides), proteins (polypeptides vs. amino acids), and nucleic acids (DNA vs. nucleotides). We noted pronounced changes in bacterial growth, activity, and transcription related to DOM characteristics. Transcriptional responses differed between compound classes, with distinct gene sets ("core genes") distinguishing carbohydrates, proteins, and nucleic acids. Moreover, we found a strong divergence in functional transcription at the level of particular monomers and polymers (i.e., the condensation state), primarily in the carbohydrates and protein compound classes. These specific responses included a variety of cellular and metabolic processes that were mediated by distinct bacterial taxa, suggesting pronounced functional partitioning of organic matter. Collectively, our findings show that two important facets of DOM, compound class and condensation state, shape bacterial gene expression, and ultimately select for distinct bacterial (functional) groups. This emphasizes the interdependency of marine bacteria and labile carbon compounds for regulating the transformation of DOM in surface waters.
Genome-wide identification of Argonautes in Solanaceae with emphasis on potato.
Z Liao, KP Hodén, RK Singh, C Dixelius
Sci Rep, 10 (1) 2045-2322 (2020)
Regulatory small RNAs (sRNAs) play important roles in many fundamental processes in plant biology such as development, fertilization and stress responses. The AGO protein family has here a central importance in gene regulation based on their capacity to associate with sRNAs followed by mRNA targeting in a sequence-complementary manner. The present study explored Argonautes (AGOs) in the Solanaceae family, with emphasis on potato, Solanum tuberosum (St). A genome-wide monitoring was performed to provide a deeper insight into gene families, genomic localization, gene structure and expression profile against the potato late blight pathogen Phytophthora infestans. Among 15 species in the Solanaceae family we found a variation from ten AGOs in Nicotiana obtusifolia to 17 in N. tabacum. Comprehensive analyses of AGO phylogeny revealed duplication of AGO1, AGO10 and AGO4 paralogs during early radiation of Solanaceae. Fourteen AGOs were identified in potato. Orthologs of AGO8 and AGO9 were missing in the potato genome. However, AGO15 earlier annotated in tomato was identified. StAGO15 differs from the other paralogs having residues of different physico-chemical properties at functionally important amino acid positions. Upon pathogen challenge StAGO15 was significantly activated and hence may play a prominent role in sRNA-based regulation of potato defense.
Long-read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore are ideal for genome-gap closure, solving structural rearrangements and sequencing through repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic-based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of DNA. Xdrop is based on isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus (HPV) 18 integrated in the genome of human HeLa cells. Analysis of the sequencing reads resolved three HPV18-chr8 integrations at base pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. Further, we enriched the complete TP53 locus in a leukemia cell line and could successfully phase co-existing mutations using PacBio sequencing. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex genomic regions. This article is protected by copyright. All rights reserved.
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