dbVar Method Types
dbVar maintains controlled vocabularies of Method Types and Analysis Types, which together are intended to capture the basic combination of steps that were followed for a given structural variation experiment. Several Analysis Types may fall under the purview of a single Method Type - for example, the Analysis Types Paired-end Mapping, Read Depth, Split Read Mapping, One End Anchored Assembly, Sequence Assembly, and SAGE all belong to the Method Type Sequencing. The following tables provide a brief description of each Method and Analysis currently allowed in dbVar submissions.
dbVar Method Types
Method Type
|
Description
|
Sequencing
|
Usually refers to high-throughput, next-generation sequencing methods, although can also refer to traditional capillary-based Sanger sequencing. Advantages: a lot of data at little cost. Disadvantages: short read length; high error rates. Next-Gen Sequencing is currently the most popular method for generating genetic data in general, and for detecting both single-nucleotide and structural variation. |
Oligo aCGH
|
The use of dense arrays of oligonucleotides deposited on glass slides, subjected to fluorescent Comparative Genomic Hybridization. Probe Intensity Analysis (see Analysis Types below) use test:reference ratios to detect copy number changes. Advantages: one can "scan" an entire genome very rapidly; high sensitivity. Disadvantages: cannot detect inversions or complex rearrangements; cannot determine genomic location of copy number gains (and in some cases losses); resolution not as high as sequencing. Another very popular method for detecting structural variation. |
SNP Array
|
Originally designed to genotype single nucleotide variation in a massively parallel manner, SNP arrays have many of the same advantages and disadvantages as oligonucleotide arrays. However, they have the added benefit of enabling statistical analysis of allelic inhertance patterns between samples from related individuals, thus adding to their ability to detect and describe de novo structural variation events. |
BAC aCGH
|
BAC arrays pre-date oligo arrays and use as probes replicated bacterial artificial chromosome DNA (average insert length ~150 kilobases) and comparative genomic hybridization between a reference and a test sample. Disadvantages: labor-intensive to produce; very low resolution. Not typically in current use. |
Curated
|
The manual aggregation and curation of independently reported structural variants from several online resources. These often clinically important variants would not otherwise find their way into a public catalogue of important structural variation. Examples include variants sporadically reported in the scientific literature, or stored in clinically-oriented online resources (e.g., OMIM™) or in locus-specific databases (LSDBs). |
Digital Array
|
The use of microfluidics and highly parallel real-time PCR to digitally count copy number in a sample. Similarly to other array-based techniques, uses comparative genomic hybridization. Rarely used, usually as a validation method. |
FISH
|
The use of multiple, differentially colored fluorescently-labeled probes (usu. fosmids, cosmids, or BACs) hybridized to a chromosome spread to detect copy number and position of target loci. Advantages: gives unambiuous relative positional information, can detect balanced and unbalanced translocations. Disadvantages: very low resolution, low throughput, labor intensive. |
Gene Expression Array
|
Similar to other array-based methods, except probes represent transcribed regions rather than the entire genome; therefore, subsequent intensity analysis reflects changes in gene expression which do not necessarily reflect changes in genomic DNA. Advantages: good for identifying possible gene deletions as a by-product of a gene expression experiment. Disadvantages: reduction or loss of signal does not necessarily indicate gene deletion - results must be followed up with more experiments. |
Karyotyping
|
May refer to any of several methods involving preparation of chromosome spreads and capable of detecting cytogenetic changes under a microscope. Examples are G-banding and fluorescence-based FISH, or chromosome "painting." |
MAPH
|
Multiplex Amplifiable Probe Hybridization; based on the quantitative recovery of short amplifiable probes after hybridization to genomic DNA. Advantages: good at detecting specific deletions in clinical samples; potential for high throughput. Rarely used. |
MassSpec
|
MOLDI-TOFF MassSpectroscopy of allele-specific primer extension products. Can be used to determine allele dosage ratios at specific loci in samples with heterozygous genotypes. Used rarely, as a validation method. |
Merging
|
Used for combining the output of multiple experiment_ids from the EXPERIMENTS tab. For example, if you only wanted to submit calls that were discovered independently by two or more experimental approaches, you could: 1. List the primary experiments in EXPERIMENTS tab, giving each a unique experiment_id; 2. Create a new experiment with method_type and analysis_type = 'Merging' to indicate that data from primary experiments was merged; 3. List the experiment_ids of the experiments that were merged, in the merged_experiment_ids field of the new experiment; and 4. Use the newly created experiment_id when reporting calls in the VARIANT CALLS tab. |
Multiple Complete Digestion
|
Fosmids or other library inserts are fully digested with multiple restriction enzymes, and the resulting restriction pattern is compared to what is expected based on a reference. Used rarely, as a validation method. |
MLPA
|
Multiplex Ligation-dependent Probe Amplification; fluorescence- and capillary-based detection of copy number changes at specific loci using multiplex PCR. Advantages: low cost; not labor-intensive; excellent for detecting specific small copy number changes like exon duplications and deletions. |
Optical Mapping
|
An integrated set of methods and analyses which involve stretching DNA into a single filament, immobilizing it on a charged surface, complete digestion with a single restriction enzyme, optical observation of the resulting restriction pattern, and comparison of results against the expected restriction pattern based on a reference genome. Advantages: ability to detect location and nature of insertions, deletions, and inversions. Disadvantages: highly specialized; cannot alone determine the sequence content or genomic origin of abberant events. |
PCR
|
The use of polymerase chain reaction technology to determine the presence or absence of a specified genomic locus. Advantages: easy; quick; inexpensive. Disadvantages: low throughput; low resolution. Typically used to genotype specific loci of interest. |
qPCR (Real-time PCR)
|
Similar to PCR but fluorescence-based and able to detect copy number with some degree of accuracy. |
ROMA
|
Representational Oligonucleotide Microarray Analysis. Same as oligo aCGH except restriction digest and PCR amplification are applied to samples to reduce genomic complexity and thereby increase hybridization efficiency. |
dbVar Analysis Types
Analysis Type
|
Description
|
Associated Method Type
|
Paired-end Mapping
|
The mapping of paired sequence reads from opposite ends of an insert; sequences are mapped to a reference genome and assayed for compared vs. expected interval. Advantages: excellent for detecting insertions and deletions; very high throughput. Disadvantage: cannot identify the sequence content of ovserved structural variation events - additional sequencign experiments are required. |
Sequencing |
Read Depth
|
Sequence-based but produces data similar to array-based methods, in that the relative representation of a given sequence indicates its copy number in the test sample. |
Sequencing |
One End Anchored Assembly
|
The analysis of paired-end reads in which only one of the two reads maps to the reference assembly. Advantages: interesting because results are novel sequences. Disadvantages: does not tell much about the structural variation event without follow-up sequencing experiments to determine size and content. |
Sequencing |
Split Read Mapping
|
Mapping of sequence reads in which detection of the variant is based on the unexpected composition of a single read - e.g., a read is "split" such that each of the resulting fragments maps to a different location in the reference assembly. Advantages: results are explicit, with basepair resolution. Disadvantages: is limited in the types of events it can detect; is hit-or-miss rather than systematic. |
Sequencing |
Sequence Assembly (de novo or local)
|
Independent assembly of a group of test sequence traces, followed by the comparison of those results against a reference assembly. Many assembly algorithms exist, as this is a rapidly-developing field (look up"Assemblathon" in the literature). The specific assembler must be indicated in the "detection_method" field of a dbVar submission. |
Sequencing |
Sequence Alignment
|
The discernment of structural variant boundaries based on the alignment of test sequence against a reference assembly. |
Sequencing |
Probe Signal Intensity
|
Computational and statistical analyses of probe signal strength reported by array-based experiments. The primary analysis for most array-based methods. Advantages: high sensitivity. |
Oligo aCGH, SNP array, BAC aCGH, FISH, qPCR |
SNP Genotyping Analysis
|
The integrated analysis of probe intensity data and inheritance patterns based on SNP array results. Although SNP arrays were not originally designed with the detection of structural variation in mind, it soon became apparent that the combination of probe intensity and allele inheritance data they produced were excellent at identifying copy number changes. |
SNP array |
MCD Analysis
|
Analysis of observed vs. expected restriction patterns based on experiments of Method Type 'Multiple Complete Digestion' (see above). |
Multiple Complete Digestion |
Merging
|
See Method Type 'Merging' above |
Merging |
Optical Mapping
|
See Method Type 'Optical Mapping' above |
Optical Mapping |
Serial Analysis of Gene Expression
|
Sequence-based assessment of gene expression patterns as determined by high-throughput capture of concatenated sequence tags derived from mRNA transcripts. Results describe patterns of gene expression which do not necessarily reflect underlying structural variation in DNA. |
Sequencing |
Manual Observation
|
Manual observation |
n/a |
Curated
|
See Method Type 'Curated' above |
Curated |
Other
|
Other |
n/a |
