Journal: bioRxiv

Article Title: Endogenous CRISPR arrays for scalable whole organism lineage tracing

doi: 10.1101/501551

Figure Lengend Snippet: Protocols for validation of CRISPR targets in vitro in Mouse cell lines (a) or in vivo in Zebrafish embryos (b). Examples of endogenous CRISPR arrays from Mouse (c) and Zebrafish (d). The Primer3 PCR primer sequences and 5’Gs of target sites are shown in bold. The CRISPR targets sites are shown in Blue. The PAM sequences are highlighted in red. e and f) Fragment size predictions from surveyor nuclease assay are shown for the two tested endogenous CRISPR arrays. The band at the higher molecular weight is the uncut amplicon and the two bands at lower molecular weight are the cut fragments. g and h) Gels from a high sensitivity DNA chip after application of the surveyor nuclease assay. Bands are of the expected size for all targets with sufficient signal. i and j) Indel detection using Miseq deep sequencing of the pooled Mouse and Zebrafish amplicons. Purple lines show the number of indels (center point of indel and averaged over a window length of 5bp) detected at that specific position in the amplicon. Vertical dashed lines represent the expected positions of indels.

Article Snippet: Furthermore longer read sequencing technologies such as Pacbio and Oxford nanopore will result in deeper and broader lineaging dendrograms through the inclusion of more CRISPR sites and thus the generation of more bits of information.

Techniques: Biomarker Discovery, CRISPR, In Vitro, In Vivo, Nuclease Assay, Molecular Weight, Amplification, Sequencing

Journal: bioRxiv

Article Title: Endogenous CRISPR arrays for scalable whole organism lineage tracing

doi: 10.1101/501551

Figure Lengend Snippet: a) Schematic showing the approach to perform lineage tracing in the zebrafish. b) Histogram of the maximum depth of the reads in the dendrogram. c) Sub-branch of the dendrogram built using indel spectrum information of amplicons after deep sequencing (See Methods). Red blocks indicate a deletion and blue blocks indicate an insertion with the size of the block representing the size of the indel. d) One sub-branch of the dendrogram is highlighted in higher resolution.

Article Snippet: Furthermore longer read sequencing technologies such as Pacbio and Oxford nanopore will result in deeper and broader lineaging dendrograms through the inclusion of more CRISPR sites and thus the generation of more bits of information.

Techniques: Sequencing, Blocking Assay

Journal: Royal Society Open Science

Article Title: Concurrent loss of ciliary genes WDR93 and CFAP46 in phylogenetically distant birds

doi: 10.1098/rsos.230801

Figure Lengend Snippet: Concurrent loss of CFAP46 gene with WDR93 . A blast-based search of genomes of representative species (shown on the x -axis of each dot plot) using the entire CFAP46 genic region, along with the 50 kb flanking sequence of the mallard ( Anas platyrhynchos ) as a query (shown on the y -axis of each dot plot), was used to generate dot plots using the xsltproc utility from XSLT sandbox ( https://github.com/lindenb/xslt-sandbox ). Conserved orthologous regions with no segmental deletions are found in ( a ) ostrich ( Struthio camelus ), ( b ) zebra finch ( Taeniopygia guttata ), ( c ) ruddy duck ( Oxyura jamaicensis ) and ( d ) black swan ( Cygnus atratus ). However, alignment patterns consistent with a segmental deletion were found in the galliform lineage in species such as ( e ) chicken ( Gallus gallus ), ( f ) helmeted guineafowl ( Numida meleagris ), ( g ) common pheasant ( Phasianus colchicus ) and ( h ) Japanese quail ( Coturnix japonica ). Similarly, in geese lineage, another segmental deletion is common to species such as ( i ) pink-footed goose ( Anser brachyrhynchus ), ( j ) swan goose ( Anser cygnoides ), ( k ) bar-headed goose ( Anser indicus ) and ( l ) Canada goose ( Branta canadensis ). Other phylogenetically independent bird species, namely ( m ) rifleman ( Acanthisitta chloris ), ( n ) ruff ( Calidris pugnax ), ( o ) Anna's hummingbird ( Calypte anna ) and ( p ) speckled mousebird ( Colius striatus ) have lost the CFAP46 gene through segmental deletions.

Article Snippet: We confirmed the correctness of the genome assembly using PacBio long-read sequencing in chicken ( Gallus gallus ) and mallard ( Anas platyrhynchos ) (see electronic supplementary material, figures S33 and S34).

Techniques: Sequencing