Biology Reference
In-Depth Information
3.3. Sequencing
Sequencing is performed using either of the primers used for PCR. Prior to
sequencing, PCR products must be purified to remove un-incorporated reaction
components. This may be performed in-house using commercially available
column-based methods or else by newer methods that utilize magnetic silica-
based technology. As a result of cost, speed, and quality, the sequencing of
PCR amplicons is frequently out-sourced to university-based core facilities or
commercial laboratories where the cost ranges from 3-12 dollars per sequence.
Some facilities currently include purification of PCR products as part of a
discounted package for high-throughput sequencing; in this case, 20 μL of
reaction product may be transferred to a new microtiter plate after verification
of amplification and shipped according to the facility's specifications (
see
Fig. 3C
).
Following retrieval of sequence data, trace chromatograms should be
inspected for each sequence to ensure quality; individual samples may
also be sequenced in both directions for purposes of confirmation.
As individual
spa
repeats are defined by single nucleotide polymor-
phisms, sequences with high background or ambiguities should not be
utilized for
spa
-typing analysis. Good-quality sequences may be submitted
directly for
spa-
typing analysis using any of several available software
platforms (e.g., eGenomics: http://www.egenomics.com, New York; Ridom:
http://www.ridom.de, Würzburg, Germany); results are returned in text-based
format that is highly amenable to personalized database management. New or
atypical
spa
types should be confirmed by sequencing in the other direction,
especially those that include novel repeats.
3.4. Analysis/Interpretation
As the main source of variation within the polymorphic X region of the
spa
gene seems to be driven by duplication or deletion of the repeat units, strain
lineages cannot be constructed by direct sequence comparison using standard
alignment algorithms
(10)
. As such, visual depictions of strain typing results
using dendrograms are precluded as they rely primarily on sequence alignment.
Therefore, to examine strain relatedness, all possible variations of the repeat
units are first identified, following which the organization of the repeats in the
polymorphic X region is compared between different isolates. In this manner,
each
spa
type denotes a collection of specific repeat units organized in a
particular pattern to the exclusion of other known types. Other algorithms to
denote relatedness of
spa
types have been developed, such as based upon repeat
pattern (BURP)
(34)
, a method that is similar to based upon related sequence
types (BURST) that is used for MLST analysis
(35)
.
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