Biomedical subjects

Allen D Delaney

Publications and source records attributed to Allen D Delaney.

4 recordsLinked to original sources

PubMed2006

Large-scale production of SAGE libraries from microdissected tissues, flow-sorted cells, and cell lines.

We describe the details of a serial analysis of gene expression (SAGE) library construction and analysis platform that has enabled the generation of >298 high-quality SAGE libraries and >30 million SAGE tags primarily from sub-microgram amounts of total RNA purified from samples acquired by microdissection. Several RNA isolation methods were used to handle the diversity of samples processed, and various measures were applied to minimize ditag PCR carryover contamination. Modifications in the SAGE protocol resulted in improved cloning and DNA sequencing efficiencies. Bioinformatic measures to automatically assess DNA sequencing results were implemented to analyze the integrity of ditag structure, linker or cross-species ditag contamination, and yield of high-quality tags per sequence read. Our analysis of singleton tag errors resulted in a method for correcting such errors to statistically determine tag accuracy. From the libraries generated, we produced an essentially complete mapping of reliable 21-base-pair tags to the mouse reference genome sequence for a meta-library of approximately 5 million tags. Our analyses led us to reject the commonly held notion that duplicate ditags are artifacts. Rather than the usual practice of discarding such tags, we conclude that they should be retained to avoid introducing bias into the results and thereby maintain the quantitative nature of the data, which is a major theoretical advantage of SAGE as a tool for global transcriptional profiling.

Animals↗

PubMed2006

Oligonucleotide microarray analysis of genomic imbalance in children with mental retardation.

The cause of mental retardation in one-third to one-half of all affected individuals is unknown. Microscopically detectable chromosomal abnormalities are the most frequently recognized cause, but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause. Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants. We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis, by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays. We found de novo deletions as small as 178 kb in eight cases, de novo duplications as small as 1.1 Mb in two cases, and unsuspected mosaic trisomy 9 in another case. This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation.

Child↗

PubMed2006

Sequence biases in large scale gene expression profiling data.

We present the results of a simple, statistical assay that measures the G+C content sensitivity bias of gene expression experiments without the requirement of a duplicate experiment. We analyse five gene expression profiling methods: Affymetrix GeneChip, Long Serial Analysis of Gene Expression (LongSAGE), LongSAGELite, 'Classic' Massively Parallel Signature Sequencing (MPSS) and 'Signature' MPSS. We demonstrate the methods have systematic and random errors leading to a different G+C content sensitivity. The relationship between this experimental error and the G+C content of the probe set or tag that identifies each gene influences whether the gene is detected and, if detected, the level of gene expression measured. LongSAGE has the least bias, while Signature MPSS shows a strong bias to G+C rich tags and Affymetrix data show different bias depending on the data processing method (MAS 5.0, RMA or GC-RMA). The bias in the Affymetrix data primarily impacts genes expressed at lower levels. Despite the larger sampling of the MPSS library, SAGE identifies significantly more genes (60% more RefSeq genes in a single comparison).

Animals↗

PubMed2005

A mouse atlas of gene expression: large-scale digital gene-expression profiles from precisely defined developing C57BL/6J mouse tissues and cells.

We analyzed 8.55 million LongSAGE tags generated from 72 libraries. Each LongSAGE library was prepared from a different mouse tissue. Analysis of the data revealed extensive overlap with existing gene data sets and evidence for the existence of approximately 24,000 previously undescribed genomic loci. The visual cortex, pancreas, mammary gland, preimplantation embryo, and placenta contain the largest number of differentially expressed transcripts, 25% of which are previously undescribed loci.

Alternative Splicing↗