long read sequencing services  (Plasmidsaurus)

Journal: bioRxiv

Article Title: Redesigning the Drosophila histone gene cluster: An improved genetic platform for spatiotemporal manipulation of histone function

doi: 10.1101/2024.04.25.591202

Figure Lengend Snippet: A) Diagram depicting histone gene clusters in H. sapiens , D. rerio , D. melanogaster , X. laevis , and C. elegans . Only chromosomes containing histone gene clusters are shown, and histone gene clusters are represented by orange boxes for all organisms except Drosophila , where the histone gene cluster is represented by a green box. B) D. melanogaster possesses ∼100 tandem-repeats of a nearly identical, 5kb histone gene unit containing each of the five replication-dependent histone genes: H1 , H2b , H2a , H4 , and H3 . C) Multiple sequence protein alignment of the five replication-dependent histones from each of the listed organisms. Amino acids are colored by identity (Hydrophobic = light green, Large Hydrophobic = dark green, Polar = purple, Small Alcohol = blue, Positive = red) and gaps are represented as grey boxes. Average amino acid similarity and identity were calculated for each species compared to H. sapiens .

Article Snippet: Plasmids were verified using Oxford Nanopore long read sequencing services by Plasmidsaurus, Inc. (Eugene, OR) including preliminary annotation by pLann.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Redesigning the Drosophila histone gene cluster: An improved genetic platform for spatiotemporal manipulation of histone function

doi: 10.1101/2024.04.25.591202

Figure Lengend Snippet: A) Diagram of the ΔHisC locus with locations of the CRISPR-Cas9 guideRNA target sequences used for gap repair. ΔHisC is marked by mini-white ( white ) with an FRT site integrated at the first intron. Breakpoints between endogenous sequence and P-element sequence are noted using dm6 coordinates ( Hoskins et al . 2015 ). The centromere-proximal breakpoint results in removal of the transcription start site and upstream regulatory region plus 26 bp of the 5’UTR of lamp1 . B) A simplified diagram of the CRISPR-Cas9 repair template plasmid, noting the distal and proximal homologies and desired insertion sequence. C) A simplified diagram of the repaired locus with Sanger sequence traces over the mutated guideRNA sequences and junctions between endogenous sequence (distal/proximal homologies) and inserted sequences (B2, and 5’-UTR of lamp1 ). D) Oxford Nanopore long read sequence validation of the ΔHisC cadillac locus. Read depth at each position of the locus is represented on the y-axis with the color indicating the percent of reads with the expected sequence. The red positions (a), (b), (c), (d), and (e) represent differences between ΔHisC cadillac and the reference sequence. E) Fully annotated diagram of the ΔHisC cadillac locus.

Article Snippet: Plasmids were verified using Oxford Nanopore long read sequencing services by Plasmidsaurus, Inc. (Eugene, OR) including preliminary annotation by pLann.

Techniques: CRISPR, Sequencing, Plasmid Preparation, Biomarker Discovery

Journal: bioRxiv

Article Title: Redesigning the Drosophila histone gene cluster: An improved genetic platform for spatiotemporal manipulation of histone function

doi: 10.1101/2024.04.25.591202

Figure Lengend Snippet: A) Diagram of the predicted integration via ΦC31 recombination between the ΔHisC cadillac attP sites and the attB site on the BAC vector. B) Diagram of resulting locus after integration at both the distal and proximal attP sites. PCR target sites spanning the junction between the integrated histone gene array/vector sequence and ΔHisC cadillac sequence are indicated in with pink bars. C) Representative DNA agarose gels with bands indicating amplification across a vector / ΔHisC cadillac junction. Summary table (right) describes the proximal and/or distal integration of different histone array multimers, the number of independent lines isolated, and (*) whether the integrated transgene supported viability of ΔHisC cadillac {Histone Array} homozygotes. “Yes**” indicates that 1 of 3 independent lines is homozygous viable. D) Nanopore validation of a pMultiBAC-6xDWT (top) and the resulting ΔHisC cadillac {D-6xDWT}, {P-6xDWT} (bottom) transformant. Grey lines indicate aligned long read data from plasmid (top; Plasmidsaurus, Inc. (Eugene, OR)) or transformants (bottom; in house). Both sets of data are aligned to the predicted ΔHisC cadillac {D-6xDWT}, {P-6xDWT} sequence (middle) including genomic DNA (blue line), pMultiBAC (grey boxes), DWT repeat (green arrows), and dsRed (from ΔHisC cadillac , red arrowed box). Anchor points for the genomic long read alignment are indicated by asterisks (*) on the cartoon reference genome.

Article Snippet: Plasmids were verified using Oxford Nanopore long read sequencing services by Plasmidsaurus, Inc. (Eugene, OR) including preliminary annotation by pLann.

Techniques: Plasmid Preparation, Sequencing, Amplification, Isolation, Biomarker Discovery

Journal: bioRxiv

Article Title: Redesigning the Drosophila histone gene cluster: An improved genetic platform for spatiotemporal manipulation of histone function

doi: 10.1101/2024.04.25.591202

Figure Lengend Snippet: A) Commercial plasmid sequencing services rely on the random circularization of plasmids prior to Oxford Nanopore sequencing. De novo assembly of these sequences collapse the repeated histone array units (green) while leaving the BAC vector (purple arrows and black line) well annotated. Sequence alignment with ClustalW (or similar) will result in gaps or masking of histone array repeats. B) Schematic of a strategy to determine the histone gene array length and sequence identity using commercial plasmid sequencing with Oxford Nanopore technology. An accurate, linearized reference sequence is generated with repeat regions (green) flanked by unique vector sequence (purple arrows and black line). The first 50-60 bp are selected as the reference origin sequence. A BLAT search is performed against every read, searching for the reference origin sequence. On every read, sequence 5’ of the identified reference origin sequence is moved to the end of the read. The modified reads are aligned, and an accurate reference sequence is generated. C) Oxford Nanopore sequencing will detect prokaryotic DNA methylation as a mis-called base. Logograms showing the depth of base calls around dcm methylation sites (Top) and EcoKI methylation sites (Bottom) grown in either DH5a (dcm+/EcoKI+) or Stellar (dcm-/EcoKI-) E. coli cell lines. D) Schematic of a strategy to determine the array length and sequence identity of integrated histone gene arrays using Oxford Nanopore sequencing. An accurate reference sequence of the target locus is generated and ∼50 bp of sequence flanking the integrated histone arrays are selected (yellow highlights). A BLAT search is performed against every read, searching for the unique reference sequences. Reads without these sequences are removed. Alignment with minimap2 generates an accurate map of the sequence.

Article Snippet: Plasmids were verified using Oxford Nanopore long read sequencing services by Plasmidsaurus, Inc. (Eugene, OR) including preliminary annotation by pLann.

Techniques: Plasmid Preparation, Sequencing, Nanopore Sequencing, Generated, Modification, DNA Methylation Assay, Methylation

Journal: bioRxiv

Article Title: Redesigning the Drosophila histone gene cluster: An improved genetic platform for spatiotemporal manipulation of histone function

doi: 10.1101/2024.04.25.591202

Figure Lengend Snippet: A) Diagram of the predicted 5 kb excision from ΔHisC cadillac after B3 recombinase expression. B) Diagram of the predicted 5 kb excision from ΔHisC cadillac after B2 recombinase expression. C) Confocal images of DAPI-stained Drosophila third instar wing imaginal discs in which UAS-B3 is expressed in the anterior compartment by Gal4 under the control of the cubitus interruptus (ci) promoter. UAS-sfGFP is also expressed in the anterior compartment and marks cells with recombinase expression. Loss of dsRed signal indicates excision of the ∼5kb dsRed expression cassette from ΔHisC cadillac . D) As in C except with expression of the B2 recombinase. E) Diagram depicting integration of a 6x wild-type histone gene array into the distal attP site of ΔHisC cadillac , location of the 41kb sequence excised upon expression of B2R is noted. F) Confocal images of two DAPI-stained Drosophila eye imaginal discs in which UAS-B2 recombinase is expressed in the full eye disc and part of the attached antennal disc by Gal4 under the control of the eyeless (ey) promoter. Loss of dsRed signal indicates excision of the 41kb dsRed expression cassette plus the 6x wild-type histone array from ΔHisCcadillac .

Article Snippet: Plasmids were verified using Oxford Nanopore long read sequencing services by Plasmidsaurus, Inc. (Eugene, OR) including preliminary annotation by pLann.

Techniques: Expressing, Staining, Control, Sequencing