March 2007, SoftGenetics
- Sequence Assembly and Mutation
Detection with Mutation Surveyor®:
Introduction
Within the past decade, shotgun sequencing
has been demonstrated to be a powerful tool in sequencing
the genomes of several organisms (1). Dueto
limitations of only several hundred consecutive bases with
adequate resolution, techniques needed to be developed to
piece together billions ofbases into consecutive order.
There are several emerging technologies that make sequencing
faster and generate short sequences. 454 LifeSciences™
utilizes the pyrosequencing technique and generates sequences
that are about 100 bases in length (2),
Illumina®’s sequencing-by-synthesismethod yields
read lengths of about 25 base pairs (3),
and the sequencing-by-ligation technique yields fragments
about 20 base pairs in length (4).The genomes
from many organisms including Drosophila melanogaster, Escherichia
coli, mouse and of course human have been fully sequenced
andassembled and are available in multiple databases for
use by the public. However, many researchers are not working
with sequences that have beenassembled and published.
For the genetic analysts working with sequences that do
not have genomic references, Mutation Surveyor is a powerful
tool capable of taking a groupof sequences and assembling
them into one long reference file. Just as the shotgun sequencing
method when used to sequence the human genomerequired these
short sequences to be assembled, many other projects require
the same functionality. Mutation Surveyor is capable of
assemblingsequences of many formats including *ab1, *abi
and *scf. The resulting text file containing a single nucleotide
text string can then be utilized directlyby the software
as the reference file or annotated similarly to a GenBank
file for identification of the coding sequence, variations
and more.
In addition to its short sequence assembly tool, Mutation
Surveyor is a powerful genetic analysis package capable
of comparing samples to referencesequences and easily finding
nucleotide and amino acid variations. All types of single
nucleotide polymorphisms (SNPs) including homozygoussubstitutions,
heterozygous substitutions with as little as 5-10% contribution
from the minor allele and insertions and deletions (INDELs)
can bedetected with the click of only a couple buttons.
Mutation Surveyor provides an accuracy >99% in detecting
homozygous and heterozygous SNPs (5), when
both forward and reverse traces are of Phred 20 quality.
In addition, the complex heterozygous INDELs are deconvoluted
from each otheryielding two clean traces for easy identification
of nucleotide variations.
For more details, download the Sequence
Assembly and Mutation Detection with Mutation Surveyor
PDF 
(150 Kb)
