Journal: Nature Communications

Article Title: Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture

doi: 10.1038/s41467-023-36099-7

Figure Lengend Snippet: a Principle of alpha-2-macroglobulin (A2M)-based protease trapping. Cleavage of the bait sequence leads to a structural rearrangement in A2M that enables reaction of cysteine-glutamine (Cys-Gln) thioester with surface lysines of the protease, thereby trapping it covalently. The bait sequence (depicted as a blue line) can be replaced by a protease cleavage site of choice. b Setup of turnover selection for libraries of yeast-displayed protease. Proteases (blue pacman) are displayed on the surface of yeast cells via an Aga2 fusion (which attaches to the yeast cell surface via disulphide bonds to Aga1; indicated by S-S); optical labels are incorporated for simultaneous quantification of protease display level and catalytic turnover, together providing a measurement of normalised activity: (i) The level of protease display is read-out via a fluorescein isothiocyanate (FITC)-conjugated anti-myc tag antibody (Y-shaped antibody symbol with green label); (ii) Protease activity is probed by incubating yeast cells with biotin-tagged A2M. The captured A2M molecules are subsequently labelled with Phycoerythrin (PE)-streptavidin (SA), which binds the biotin moiety. Optionally, the signal may be amplified through a PE-anti-streptavidin antibody. c A2M cap -based detection of protease activity via flow cytometry. SplB-displaying yeast cells were reacted with A2M-WELQ ↓ S or A2M-ENLYFQ ↓ S (cognate recognition sequence of TEV protease, ENLYFQ ↓ S, serves as negative control; low activity of SplB expected towards this substrate). SplB-S157A is a mutant that is displayed on yeast, but inactive as a protease ( n = 1 flow cytometry measurement). Source data are provided as a Source Data file for c .

Article Snippet: Sequencing of the input library using circular consensus sequencing with Oxford Nanopore technology (Fig. ) , revealed the presence of 13,994 different Spl chimeras (62% of the theoretically possible combinations) and an acceptable overall recombination preference (the most overrepresented variant was 18-fold above the median read-count) (Fig. ).

Techniques: Sequencing, Selection, Activity Assay, Amplification, Flow Cytometry, Negative Control, Mutagenesis

Journal: Nature Communications

Article Title: Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture

doi: 10.1038/s41467-023-36099-7

Figure Lengend Snippet: a Schematic of selection strategy for SCHEMA-shuffled proteases using three different substrates and nanopore-based sequencing ( b ) Sequence analysis of input and output populations. Within each graph, sequences of shuffled variants are depicted as lines coloured by block ID at each position. The number of mappable INC-seq reads is shown for each condition/population. c Flow cytometry of input and output populations probed with the three different A2M substrates used during selection (representative data of n = 2 independent inductions and flow cytometry measurements for 1st round and input library, n = 1 for 2nd round). RCA rolling circle amplification, PCR polymerase chain reaction, MACS magnetic activated cell sorting, FITC Fluorescein isothiocyanate, Strep-PEStreptavidin-Phycoerythrin. Source data is provided as a Source Data file for b and c .

Article Snippet: Sequencing of the input library using circular consensus sequencing with Oxford Nanopore technology (Fig. ) , revealed the presence of 13,994 different Spl chimeras (62% of the theoretically possible combinations) and an acceptable overall recombination preference (the most overrepresented variant was 18-fold above the median read-count) (Fig. ).

Techniques: Selection, Nanopore Sequencing, Sequencing, Blocking Assay, Flow Cytometry, Amplification, Polymerase Chain Reaction, FACS

Journal: Current opinion in insect science

Article Title: Best Practices in Insect Genome Sequencing: What Works and What Doesn’t

doi: 10.1016/j.cois.2015.02.013

Figure Lengend Snippet: De novo genome assembly strategies Assembly software is designed for a specific sequencing and assembly strategy. Thus sequence must be generated with the assembly software and algorithm in mind, choosing a sequence strategy designed for a different assembly algorithm, or sequencing without thinking about assembly is usually a recipe for poor unpublishable assemblies [ 38 ]. Here we survey different assembly strategies, with different sequence and library construction requirements. A typical genome project starts with high quality DNA of as low polymorphism as available, and extends beyond genome assembly to include gene annotation. Relatively inexpensive RNAseq from multiple tissues/or life stages (the authors often chooses adult male, adult female and mixed other life stages) provides transcript data for final genome annotation. For the definition of sex chromosomes, it is often useful to re-sequence at 30X coverage one individual of each sex. Additionally, re-sequencing of individuals at 30X genome coverage followed by alignment to the final reference using standard human analysis tools is the best way to characterize sequence variation within a species.

Article Snippet: PacBio Self-correction Overlap-layout-consensus Read sizes, coverage 6–15kb reads at 60x Open in a separate window HBAR/Falcon & Celera assembler ++++ +++ All-against-all read alignment for error correction is processing intensive PacBio Circular Consensus Sequencing Overlap-layout-consensus Reads sizes, coverage 3kb CCS reads, 20X Open in a separate window Celera assembler ++ +++ Trivial error correction to Sanger quality long reads.

Techniques: Software, Sequencing, Generated, Scaffolding

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Human-specific tandem repeat expansion and differential gene expression during primate evolution

doi: 10.1073/pnas.1912175116

Figure Lengend Snippet: STR/VNTR sequence composition plots. The 4 loci represent STRs/VNTRs with ≥40 tandem repeat copies. The sequences from each human and NHP haplotype were colored according to their k-mer abundance (Materials and Methods). For the CHM13 sample, sequences from both CLR and HiFi assemblies have been included labeled as “CHM13” and “CHM13_HiFi,” respectively, which provide a replicate measure of sequence accuracy. (A) An STR located upstream of RNF219 is composed of 7 to 62 uninterrupted tandem copies of an AAAG expansion in humans. Three human haplotypes contain clustered AAGG interruptions, while 1 chimpanzee haplotype contains a clustered interruption of AG repeats. (B) A human-specific STR expansion is located in the intron of PHLDB2 and is composed of 18 to 70 uninterrupted repeats of AAG. Periodic interruptions of AGG exist in 3 human haplotypes and GRCh38. (C) An STR located in the intron of ADGRE2 contains 3 to 42 uninterrupted tandem repeat copies of TTTC. A cluster of a continuous tract of pure TC repeats interrupts the tetranucleotide repeat in 1 of the Puerto Rican haplotypes. (D) A human-specific VNTR expansion is located in the intron of CLCN5 and is composed of 21 to 80 tandem repeat copies of a 26-bp motif. A single interruption by a 30-bp motif that contains 3 additional adenines in position 23 occurs in the Puerto Rican and Yoruban haplotypes (gray bars).

Article Snippet: 3) Validation with orthogonal long-read sequence datasets: we carried out an additional orthogonal validation for STRs and VNTRs from the CHM13 continuous long-read (CLR) assembly using orthogonal high-fidelity (HiFi) circular consensus sequencing data and ultralong Oxford Nanopore Technologies (UL-ONT) sequence reads generated from the same source cell line (CHM13) ( 29 ).

Techniques: Sequencing, Labeling

Journal: Wellcome Open Research

Article Title: The genome sequence of the small pearl-bordered fritillary butterfly, Boloria selene (Schiffermüller, 1775)

doi: 10.12688/wellcomeopenres.17734.1

Figure Lengend Snippet: Dorsal (left) and ventral (right) surface view of wings from specimen UK_BS_1216 (ilBolSele5) from Carrifran Wildwood, Scotland, used to generate 10X, HiFi and Hi-C data.

Article Snippet: Pacific Biosciences HiFi circular consensus (for ilBolSele5) and 10X Genomics read cloud (ilBolSele5 and ilBolSele2) DNA sequencing libraries were constructed according to the manufacturers’ instructions.

Techniques: Hi-C