scholarly journals CRISPR-LRS for mapping transgenes in the mouse genome

2022 ◽  
Author(s):  
W. Bart Bryant ◽  
Allison Yang ◽  
Susan Griffin ◽  
Wei Zhang ◽  
Xiaochun Long ◽  
...  
Keyword(s):  
Copy Number ◽  
Mouse Genome ◽  
Bacterial Artificial Chromosomes ◽  
Noncoding Sequences ◽  
Transgenic Mouse Line ◽  
Artificial Chromosomes ◽  
Transgene Integration ◽  
Copy Numbers ◽  
Long Read ◽  
Driver Line

Microinjected transgenes, including bacterial artificial chromosomes (BACs), insert randomly in the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and the accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. Here, we introduce CRISPR-Cas9 long-read sequencing (CRISPR-LRS) to ascertain transgene integration locus and estimated copy number. This method revealed integration loci for both a BAC and Cre-driver line, and estimated the copy numbers for two other BAC mouse lines. CRISPR-LRS offers an easy approach to establish robust breeding practices and accurate phenotyping of most any transgenic mouse line.

scholarly journals Facile Recovery of Individual High-Molecular-Weight, Low-Copy-Number Natural Plasmids for Genomic Sequencing

2006 ◽  
Vol 72 (7) ◽  
pp. 4899-4906 ◽  
Author(s):  
Laura E. Williams ◽  
Chris Detter ◽  
Kerrie Barry ◽  
Alla Lapidus ◽  
Anne O. Summers
Keyword(s):  
Plasmid Dna ◽  
Copy Number ◽  
Mobile Genetic Element ◽  
Large Plasmid ◽  
Bacterial Artificial Chromosomes ◽  
High Coverage ◽  
Artificial Chromosomes ◽  
Gram Positive Bacteria ◽  
Low Copy Number ◽  
Plasmid Isolation

ABSTRACT Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of high-coverage libraries, as shown by sequencing five native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.

scholarly journals Copy Number Changes in 1q21.3 and 1q23.3 have Different Clinical Relevance in Ovarian Tumors

2009 ◽  
Vol 12 (2) ◽  
pp. 29-37 ◽  
Author(s):  
I Dimova ◽  
B Orsetti ◽  
Ch. Theillet ◽  
R. Dimitrov ◽  
D Toncheva
Keyword(s):  
Clinical Relevance ◽  
Copy Number ◽  
Tissue Microarrays ◽  
Ovarian Tumors ◽  
Specific Gene ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Copy Number Changes ◽  
Benign Ovarian Tumors ◽  
Serous Tumors

Copy Number Changes in 1q21.3 and 1q23.3 have Different Clinical Relevance in Ovarian TumorsMany studies have reported aberrations such as amplifications, deletions and translocations of 1q21-q23 in ovarian tumors. These findings increase the scientific interest in analyzing this region using specific gene probes. We investigated the frequency of copy number changes of two specific bacterial artificial chromosomes (BAC) clones in 1q21.3 and 1q23.3 by fluorescent in situ hybridization (FISH) on tissue microarrays consisting of 540 ovarian tumors of different malignancies, histology, stage and grade. Such changes in 1q21.3 were established in 9.64% of malignant (2.41% amplification), in 8.33% of low malignant potential (LMP) and in 13.13% of benign ovarian tumors. Copy number changes of 1q23.3 were found in 17.78% of malignant (1.48% amplification), in 16.67% of LMP and in 12.64% of benign ovarian tumors. We found a significantly higher gain of 1q23.3 in non epithelial (50%) compared to epithelial tumors (14.73%) (p <0.03). The gain of 1q21.3 prevailed in non serous malignant and LMP ovarian tumors in comparison to serous tumors. In non serous tumors, both gains were associated with higher grade. The frequency of gain in 1q23.3 was 2.5-times higher than that in 1q21.3 of ovarian cancers.

Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles

2003 ◽  
Vol 21 (4) ◽  
pp. 443-447 ◽  
Author(s):  
Giuseppe Testa ◽  
Youming Zhang ◽  
Kristina Vintersten ◽  
Vladimir Benes ◽  
W.W.M. Pim Pijnappel ◽  
...  
Keyword(s):  
Mouse Genome ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes

scholarly journals Evidence for opposing selective forces operating on human-specific duplicated TCAF genes in Neanderthals and humans

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
PingHsun Hsieh ◽  
Vy Dang ◽  
Mitchell R. Vollger ◽  
Yafei Mao ◽  
Tzu-Hsueh Huang ◽  
...  
Keyword(s):  
Copy Number ◽  
Homo Sapiens ◽  
Segmental Duplications ◽  
Sensor Protein ◽  
Long Read ◽  
Number Variation ◽  
Selective Forces ◽  
Structural Mutations ◽  
Cold Sensor ◽  
Human Specific

AbstractTRP channel-associated factor 1/2 (TCAF1/TCAF2) proteins antagonistically regulate the cold-sensor protein TRPM8 in multiple human tissues. Understanding their significance has been complicated given the locus spans a gap-ridden region with complex segmental duplications in GRCh38. Using long-read sequencing, we sequence-resolve the locus, annotate full-length TCAF models in primate genomes, and show substantial human-specific TCAF copy number variation. We identify two human super haplogroups, H4 and H5, and establish that TCAF duplications originated ~1.7 million years ago but diversified only in Homo sapiens by recurrent structural mutations. Conversely, in all archaic-hominin samples the fixation for a specific H4 haplotype without duplication is likely due to positive selection. Here, our results of TCAF copy number expansion, selection signals in hominins, and differential TCAF2 expression between haplogroups and high TCAF2 and TRPM8 expression in liver and prostate in modern-day humans imply TCAF diversification among hominins potentially in response to cold or dietary adaptations.

scholarly journals Accucopy: accurate and fast inference of allele-specific copy number alterations from low-coverage low-purity tumor sequencing data

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xinping Fan ◽  
Guanghao Luo ◽  
Yu S. Huang
Keyword(s):  
Copy Number ◽  
Bayesian Learning ◽  
Kernel Smoothing ◽  
Gaussian Mixture ◽  
Copy Number Alterations ◽  
Sequencing Data ◽  
Copy Numbers ◽  
Allele Specific ◽  
Tumor Sequencing ◽  
Low Coverage

Abstract Background Copy number alterations (CNAs), due to their large impact on the genome, have been an important contributing factor to oncogenesis and metastasis. Detecting genomic alterations from the shallow-sequencing data of a low-purity tumor sample remains a challenging task. Results We introduce Accucopy, a method to infer total copy numbers (TCNs) and allele-specific copy numbers (ASCNs) from challenging low-purity and low-coverage tumor samples. Accucopy adopts many robust statistical techniques such as kernel smoothing of coverage differentiation information to discern signals from noise and combines ideas from time-series analysis and the signal-processing field to derive a range of estimates for the period in a histogram of coverage differentiation information. Statistical learning models such as the tiered Gaussian mixture model, the expectation–maximization algorithm, and sparse Bayesian learning were customized and built into the model. Accucopy is implemented in C++ /Rust, packaged in a docker image, and supports non-human samples, more at http://www.yfish.org/software/. Conclusions We describe Accucopy, a method that can predict both TCNs and ASCNs from low-coverage low-purity tumor sequencing data. Through comparative analyses in both simulated and real-sequencing samples, we demonstrate that Accucopy is more accurate than Sclust, ABSOLUTE, and Sequenza.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

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