Dataset Information

WGA-Alexa Conjugates for Axonal Tracing.

ABSTRACT: Anatomical labeling approaches are essential for understanding brain organization. Among these approaches are various methods of performing tract tracing. However, a major hurdle to overcome when marking neurons in vivo is visibility. Poor visibility makes it challenging to image a desired neuronal pathway so that it can be easily differentiated from a closely neighboring pathway. As a result, it becomes impossible to analyze individual projections or their connections. The tracer that is chosen for a given purpose has a major influence on the quality of the tracing. Here, we describe the wheat germ agglutinin (WGA) tracer conjugated to Alexa fluorophores for reliable high-resolution tracing of central nervous system projections. Using the mouse cerebellum as a model system, we implement WGA-Alexa tracing for marking and mapping neural circuits that control motor function. We also show its utility for marking localized regions of the cerebellum after performing single-unit extracellular recordings in vivo. © 2017 by John Wiley & Sons, Inc.

SUBMITTER: Levy SL

PROVIDER: S-EPMC5830103 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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WGA-Alexa Conjugates for Axonal Tracing.

Levy Sabrina L SL White Joshua J JJ Lackey Elizabeth P EP Schwartz Lindsey L Sillitoe Roy V RV

Current protocols in neuroscience 20170410

Anatomical labeling approaches are essential for understanding brain organization. Among these approaches are various methods of performing tract tracing. However, a major hurdle to overcome when marking neurons in vivo is visibility. Poor visibility makes it challenging to image a desired neuronal pathway so that it can be easily differentiated from a closely neighboring pathway. As a result, it becomes impossible to analyze individual projections or their connections. The tracer that is chosen ...[more]

PMID: 28398642

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Project description:BackgroundGenotyping assays often require substantial amounts of DNA. To overcome the problem of limiting amounts of available DNA, Whole Genome Amplification (WGA) methods have been developed. The multiple displacement amplification (MDA) method using Φ29 polymerase has become the preferred choice due to its high processivity and low error rate. However, the uniformity and fidelity of the amplification process across the genome has not been extensively characterized.ResultsTo assess amplification uniformity, we used array-based comparative genomic hybridization (aCGH) to evaluate DNA copy number variations (CNVs) in DNAs amplified by two MDA kits: GenomiPhi and REPLI-g. The Agilent Human CGH array containing nearly one million probes was used in this study together with DNAs from a normal subject and 2 cystic fibrosis (CF) patients. Each DNA sample was amplified 4 independent times and compared to its native unamplified DNA. Komogorov distances and Phi correlations showed a high consistency within each sample group. Less than 2% of the probes showed more than 2-fold CNV introduced by the amplification process. The two amplification kits, REPLI-g and GenomiPhi, generate very similar amplified DNA samples despite the differences between the unamplified and amplified DNA samples. The results from aCGH analysis indicated that there were no obvious CNVs in the CFTR gene region due to WGA when compared to unamplified DNA. This was confirmed by quantitative real-time PCR copy number assays at 10 locations within the CFTR gene. DNA sequencing analysis of a 2-kb region within the CFTR gene showed no mutations introduced by WGA.ConclusionThe relatively high uniformity and consistency of the WGA process, coupled with the low replication error rate, suggests that WGA DNA may be suitable for accurate genotyping. Regions of the genome that were consistently under-amplified were found to contain higher than average GC content. Because of the consistent differences between the WGA DNA and the native unamplified DNA, characterization of the genomic region of interest, as described here, will be necessary to ensure the reliability of genotyping results from WGA DNA.

Project description:Amplification of minute quantities of DNA is a fundamental challenge in low-biomass metagenomic and microbiome studies because of potential biases in coverage, guanine-cytosine (GC) content, and altered species abundances. Whole genome amplification (WGA), although widely used, is notorious for introducing artifact sequences, either by amplifying laboratory contaminants or by nonrandom amplification of a sample's DNA. In this study, we investigate the effect of REPLI-g multiple displacement amplification (MDA; Qiagen, Valencia, CA, USA) on sequencing data quality and species abundance detection in 8 paired metagenomic samples and 1 titrated, mixed control sample. We extracted and sequenced genomic DNA (gDNA) from 8 environmental samples and compared the quality of the sequencing data for the MDA and their corresponding non-MDA samples. The degree of REPLI-g MDA bias was evaluated by sequence metrics, species composition, and cross-validating observed species abundance and species diversity estimates using the One Codex and MetaPhlAn taxonomic classification tools. Here, we provide evidence of the overall efficacy of REPLI-g MDA on retaining sequencing data quality and species abundance measurements while providing increased yields of high-fidelity DNA. We find that species abundance estimates are largely consistent across samples, even with REPLI-g amplification, as demonstrated by the Spearman's rank order coefficient (R2 > 0.8). However, REPLI-g MDA often produced fewer classified reads at the species, genera, and family level, resulting in decreased species diversity. We also observed some areas with the PCR "jackpot effect," with varying input DNA values for the Metagenomics Research Group (MGRG) controls at specific genomic loci. We visualize this effect in whole genome coverage plots and with sequence composition analyses and note these caveats of the MDA method. Despite overall concordance of species abundance between the amplified and unamplified samples, these results demonstrate that amplification of DNA using the REPLI-g method has some limitations. These concerns could be addressed by future improvements in the enzymes or methods for REPLI-g to be considered a >99% robust method for increasing the amount of high-fidelity DNA from low-biomass samples or at the very least, accounted for during computational analysis of MDA samples.

Dataset Information

WGA-Alexa Conjugates for Axonal Tracing.

Publications

WGA-Alexa Conjugates for Axonal Tracing.

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