Filamentous fungi of the genus Aspergillus include producers of industrially important organic acids, enzymes, and secondary metabolites, as well as pathogens of many plants and animals. Novel genes in the Aspergillus genome are potentially crucial for the fermentation and drug industries (e.g., agrochemicals and antifungal drugs). A research approach based on classical genetics is effective for identifying functionally unknown genes. During analyses based on classical genetics, mutations must be identified easily and quickly. Herein, we report the development of a cosmid-based plasmid pTOCK1 and the use of a genomic library of Aspergillus nidulans constructed using pTOCK1. The cosmid-based genomic library was used for convenient auxotrophic mutants (pyroA and pabaB), as well as mutants with abnormal colony morphology (gfsA) and yellow conidia (yA), to obtain library clones complementary to these phenotypes. The complementary strain could be obtained through a single transformation, and the cosmid could be rescued. Thus, our cosmid library system can be used to identify the causative gene in a mutant strain.

Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield approximately 106 cfu/µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 cfu/µg plasmid DNA. The protocol generates sufficient transformants for 10-100× coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries\' size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method\'s efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required.

Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screening is revolting the genetic analysis of a cellular or molecular phenotype in question but is challenged by the large size of single-guide RNA (sgRNA) library. Here we designed a minimal genome-wide human sgRNA library, H-mLib, which is composed of 21,159 sgRNA pairs assembled based on a dedicated selection strategy from all potential SpCas9/sgRNAs in the human genome. These sgRNA pairs were cloned into a dual-gRNA vector each targeting one gene, resulting in a compact library size nearly identical to the number of human protein-coding genes. The performance of the H-mLib was benchmarked to other CRISPR libraries in a proliferation screening conducted in K562 cells. We also identified groups of core essential genes and cell-type specific essential genes by comparing the screening results from the K562 and Jurkat cells. Together, the H-mLib exemplified high specificity and sensitivity in identifying essential genes while containing minimal library complexity, emphasizing its advantages and applications in CRISPR screening with limited cell numbers.

The lack of genetic tools to manipulate protozoan pathogens has limited the use of genome-wide approaches to identify drug or vaccine targets and understand these organisms\' biology. We have developed an efficient method to construct genome-wide libraries for yeast surface display (YSD) and developed a YSD fitness screen (YSD-FS) to identify drug targets. We show the efficacy of our method by generating genome-wide libraries for Trypanosoma brucei, Trypanosoma cruzi, and Giardia lamblia parasites. Each library has a diversity of ∼105 to 106 clones, representing ∼6- to 30-fold of the parasite\'s genome. Nanopore sequencing confirmed the libraries\' genome coverage with multiple clones for each parasite gene. Western blot and imaging analysis confirmed surface expression of the G. lamblia library proteins in yeast. Using the YSD-FS assay, we identified bonafide interactors of metronidazole, a drug used to treat protozoan and bacterial infections. We also found enrichment in nucleotide-binding domain sequences associated with yeast increased fitness to metronidazole, indicating that this drug might target multiple enzymes containing nucleotide-binding domains. The libraries are valuable biological resources for discovering drug or vaccine targets, ligand receptors, protein-protein interactions, and pathogen-host interactions. The library assembly approach can be applied to other organisms or expression systems, and the YSD-FS assay might help identify new drug targets in protozoan pathogens.

BACKGROUND: The genus Hypophthalmus comprises six species (H. edentatus, H. marginatus, H. fimbriatus, and H. oremaculatus), and the recently described: H. donascimientoi and H. celiae. The popular name for Hypophthalmus spp. in Brazil is mapará, this name refers to the six species. This group of fish has commercial importance for the states of Amazonas and Pará and, for this reason, requires studies to identify fish stocks. One approach is to use molecular markers, which have been very useful in studies with identification and population analysis of fish. Microsatellite molecular markers (SSRs) are one of the most informative markers for this purpose. There is little populations study of Hypophthalmus using SSRs, and there are less than six loci for the species Hypophthalmus marginatus available in the literature. With the construction of a genomic library of H. donascimientoi, we aimed to isolate and characterize SSRS markers and evaluate the extent of interspecific amplification.
RESULTS: A genomic library was constructed with regions enriched of microsatellite for Hypophthalmus donascimientoi. A total of 126 contigs with 42 SSRs were used to design flanking primers for 39 microsatellites. Fifteen loci were characterized in three locations of the Solimões/Amazonas Rivers. The number of alleles ranged from one to 17 with a total of 126 alleles. The mean observed heterozygosity (HO) and expected heterozygosity (HE) were 0.721-0.692, respectively (S.d. HO 0.061 and HE 0.060). Two loci showed significant deviation in the HWE. The PIC ranged from 0.375 to 0.908. Such identified, 12 highly informative loci, and two moderately informative loci. Among the fifteen loci characterized, seven were successfully amplified in four other species of the genus.
CONCLUSIONS: The microsatellite showed promise for estimating the genetic variability of H. donascimientoi and can be used as an efficient tool in population analyses of this species and in congeneric species analyzed.

Genome sequencing of small species, such as those of meinofauna, can be challenging due to the extremely low input of genomic DNA. While nanopore sequencing is a promising technology for genome assembly due to its limitless long reads, recommended input of 1 μg for the Ligation Sequencing Kit often precludes the use of this technology. Here, I detail an unbiased droplet-based multiple displacement amplification of picogram order of DNA to realize nanopore sequencing with ultralow input of genomic DNA. For this purpose, a microfluidic chip of 10X Genomics Chromium Controller is utilized. With this method, over 10 μg of unbiased amplicons around 10 kbp in length can be obtained from as low as 50 μg of input DNA, which is enough for the construction of multiple sequencing libraries, or for the size selection of longer DNA fragments.

BACKGROUND: Butter catfish (Ompok bimaculatus) is a preferred species in South East Asia, with huge aquaculture potential. However, there is limited information about genetic stock composition due to insufficient markers. The goal of this study was to develop de novo microsatellite markers.
RESULTS: For sequencing, genomic SMRT bell libraries (1.5 Kbp size) were prepared for O. bimaculatus. A total of 114 SSR containing sequences were used for primer designing. Polymorphic loci were validated by genotyping 83 individuals from four distant riverine populations, viz., Brahmaputra, Bichiya, Gomti and Kaveri. A total of 30 microsatellite loci were polymorphic, of which five were found to be associated with functional genes and eight (four positive and four negative) loci were found to be under selection pressure. A total of 115 alleles were detected in all loci and PIC ranged from 0.539 to 0.927 and pair-wise FST values from 0.1267 to 0.26002 (p < 0.001), with an overall FST value of 0.17047, indicating the presence of population sub-structure. Cross-species transferability of 29 loci (96.67%) was successful in congener species, Ompok pabda.
CONCLUSIONS: The novel SSR markers developed in this study would facilitate stock characterization of natural populations, to be used in future selection breeding programs and planning conservation strategies in these species. Identified non-neutral markers will give insights into the effect of local adaptation on genetic differentiation in the natural population of this species.

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Genome skimming is a popular method in plant phylogenomics that do not include a biased enrichment step, relying on random shallow sequencing of total genomic DNA. From these data the plastome is usually readily assembled and constitutes the bulk of phylogenetic information generated in these studies. Despite a few attempts to use genome skims to recover low copy nuclear loci for direct phylogenetic use, such endeavor remains neglected. Causes might include the trade-off between libraries with few reads and species with large genomes (i.e., missing data caused by low coverage), but also might relate to the lack of pipelines for data assembling.
A pipeline and its companion R package designed to automate the recovery of low copy nuclear markers from genome skimming libraries are presented. Additionally, a series of analyses aiming to evaluate the impact of key assembling parameters, reference selection and missing data are presented.
A substantial amount of putative low copy nuclear loci was assembled and proved useful to base phylogenetic inference across the libraries tested (4 to 11 times more data than previously assembled plastomes from the same libraries).
Critical aspects of assembling low copy nuclear markers from genome skims include the minimum coverage and depth of a sequence to be used. More stringent values of these parameters reduces the amount of assembled data and increases the relative amount of missing data, which can compromise phylogenetic inference, in turn relaxing the same parameters might increase sequence error. These issues are discussed in the text, and parameter tuning through multiple comparisons tracking their effects on support and congruence is highly recommended when using this pipeline. The skimmingLoci pipeline (https://github.com/mreginato/skimmingLoci) might stimulate the use of genome skims to recover nuclear loci for direct phylogenetic use, increasing the power of genome skimming data to resolve phylogenetic relationships, while reducing the amount of sequenced DNA that is commonly wasted.

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