Journal: Nutrients

Article Title: Integrated Analysis of Genomic and Genome-Wide Association Studies Identified Candidate Genes for Nutrigenetic Studies in Flavonoids and Vascular Health: Path to Precision Nutrition for (Poly)phenols

doi: 10.3390/nu16091362

Figure Lengend Snippet: Variant types, molecular consequences, most severe clinical significances, and top 10 genetic variants with highest frequencies, as well as the number of associations with pharmacologic relevance in selected n = 20 top-priority candidate genes.

Article Snippet: TCF7L2 , single nucleotide variant (846) deletion (11) insertion (9) indel (129) , missense variant (4) frameshift variant (1) synonymous variant (1) intron variant (844) 5 prime UTR variant (6) 3 prime UTR variant (8) 500 B downstream variant (5) 2 KB upstream variant (15) , drug-response (1) risk-factor (2) benign (2) , 14 , rs720785 rs7918976 rs11196171 rs11196170 rs2296784 rs720784 rs7897438 rs290476 rs10885399 rs61875109 , intron variant not specified intron variant intron variant intron variant intron variant intron variant intron variant intron variant intron variant , G,A,C C,A,G,T A,C,G G,A,C,T T,C A,C,G,T C,A,G,T G,A,C,T T,A,G C,A,G,T , G A A G T A A T A A , 0.499401 0.498602 0.495607 0.494609 0.494409 0.49401 0.478235 0.477835 0.477436 0.477236.

Techniques: Variant Assay

Journal: Integrative and Comparative Biology

Article Title: CRISPR/Cas9 Protocols for Disrupting Gene Function in the Non-vertebrate Chordate Ciona

doi: 10.1093/icb/icae108

Figure Lengend Snippet: Validation of sgRNAs for CRISPR/Cas9 in Ciona . Panel ( A ) shows schematic diagram of the protocol for validation of sgRNA mutagenesis (indel) efficacies in Ciona F0 embryos. See text and supplemental methods for detailed protocols. Panel ( B ) shows diagram of rationale for mutagenesis efficacy estimates by Illumina-based NGS. Indels represented as red blocks in target DNAs and amplicons. Different forward and reverse PCR primers (half-arrows) used to amplify targeted regions indicated in different colors. Note that, in theory, untargeted amplicons can serve as negative controls even if they are amplified from CRISPR'd embryos (see text for details).

Article Snippet: Bottom: Indel plots (deletions blue, insertions green) automatically generated by the Illumina sequencing “Amplicon-EZ” service from Azenta (Genewiz).

Techniques: Biomarker Discovery, CRISPR, Mutagenesis, Amplification

Journal: Integrative and Comparative Biology

Article Title: CRISPR/Cas9 Protocols for Disrupting Gene Function in the Non-vertebrate Chordate Ciona

doi: 10.1093/icb/icae108

Figure Lengend Snippet: Example of sgRNA validation by a commercial NGS service. Top: Diagram of Rab11-related.b gene from C. robusta ( intestinalis Type A), indicating the target locations of three candidate sgRNAs (“2.45”, “2.187”, and “3.30”, thin diagonal lines). Thicker rectangles indicate exons, thinner lines indicate introns. ATG: Translation start codon. Bottom: Indel plots (deletions blue, insertions green) automatically generated by the Illumina sequencing “Amplicon-EZ” service from Azenta (Genewiz). Control sequences were amplified from larvae electroporated with unrelated sgRNAs instead. Note different y-axis scale for each plot. Asterisks indicate natural deletions identified, due to use of genetically diverse wild Ciona populations. Total mutagenesis efficacies (automatically calculated by the service) indicated in red parentheses. For sgRNA 3.30, the efficacy rate that was automatically calculated was not reliable due to the high prevalence of a naturally occurring deletion in this particular batch of animals.

Article Snippet: Bottom: Indel plots (deletions blue, insertions green) automatically generated by the Illumina sequencing “Amplicon-EZ” service from Azenta (Genewiz).

Techniques: Biomarker Discovery, Generated, Illumina Sequencing, Amplification, Control, Mutagenesis

Journal: Integrative and Comparative Biology

Article Title: CRISPR/Cas9 Protocols for Disrupting Gene Function in the Non-vertebrate Chordate Ciona

doi: 10.1093/icb/icae108

Figure Lengend Snippet: Analysis of off-target effects (or lack thereof) in Ciona . Panel ( A ) left shows indel plot for the Dkk3.2.100 sgRNA, at its intended target (exon 2 of the Dkk3 gene, KH ID number KH.C8.904). Right: Indel profile plot zoomed in at the target site, showing a peak centered around a few basepairs 5′ to the PAM. Panel ( B ) shows one example of a candidate off-target of the Dkk3.2.100 sgRNA (KH.C9.571 intron). Top left panel shows indel plot from sequencing the KH.C9.571 intron off-target amplicon PCR-amplified from the same sample that gave the on-target plot in panel A. Top right panel shows off-target sequence and coordinates (KH genome assembly) as predicted by CRISPOR. Mismatches with the intended sgRNA (“guide”) target sequence are indicated by asterisks and bold font. The all-important sgRNA “seed” region is underlined, with the PAM in italics. Bottom panel shows zoomed-in indel profile plot for the KH.C9.571 intronic off-target site, showing that the negligible (<1%) frequency of scattered indels are not enriched at the predicted off-target PAM/seed region. Panel ( C ) shows analysis of sgRNA efficacy at a target with an alternative, non-canonical PAM for S. pyogenes Cas9 (AAG). An sgRNA (1) targeting the Irx.a gene at a sequence (bold letters) with an AAG alternative PAM (red italics) resulted in a 7% indel rate. There was no detectable indels by sgRNAs 2 and 3 targeting sequences with adjacent motifs not previously reported to function as alternative PAMs (TAC and ACC, gray italics).

Article Snippet: Bottom: Indel plots (deletions blue, insertions green) automatically generated by the Illumina sequencing “Amplicon-EZ” service from Azenta (Genewiz).

Techniques: Sequencing, Amplification