Biology Reference
In-Depth Information
where more details on sample preparation and data processing can
be found.
Materials and Methods
MASSIVELY PARALLEL SIGNATURE SEQUENCING
(
MPSS
)
A review of the principal stages of the MPSS protocol follows; more
detailed discussions are available elsewhere [3,9,10].
1.
cDNA signature/tag conjugate library construction
. Poly(A)
+
mRNA
is extracted from the tissue of interest from which cDNA is
synthesized. The 20 bases adjacent to the 3'-most
Dpn
II site
(GATC) of each cDNA are captured using a type IIs restriction
enzyme. The GATC and its contiguous 13-mer form a 17-mer
sequence referred to as a “signature.” These signatures are
then PCR amplified and cloned into a collection of 8
8
distinct
vectors (the “tags”), thus adding a unique identification tag to
each signature.
2.
Microbead loading.
Multiple pools of about 640,000 signature/
tag conjugates are amplified (i.e.,
4% of tag complexity) and,
after the tags are rendered single-stranded, these conjugates
are hybridized with microbeads, each of which has bound to
it
∼
10
4
copies of one of the 8
8
possible antitags. The 4% of
these microbeads with antitags corresponding to the tags of
the conjugates (and thus each hybridized or “loaded” with
∼
∼
10
4
copies of the signature/tag conjugate to its antitag) is iso-
lated using a fluorescence-activated cell sorter. Approximately
1.5
10
6
loaded microbeads are assembled in a flow cell and
the signature sequence on each bead is determined by MPSS.
3.
Massively Parallel Signature Sequencing.
The signatures are
sequenced by the parallel identification of four bases by
hybridization to fluorescently labeled encoders, followed by
removal of that set of four bases and exposure of the next four
bases by a type IIs endonuclease digestion. The process is then
repeated. The imaged fluorescent data are processed to yield
the number of beads that have a given signature sequence. Two
types of initiating adaptors, whose type IIs restriction sites are
offset by two bases, are ligated to two separate sets of microbeads
containing a replicate of the same signature library. This is
done to reduce signature losses from self-ligation of ends of
signatures produced when digestion exposes palindromic
overhangs. These two alternative sequencing reactions are
referred to as “2-stepper” (TS) and “4-stepper” (FS) sequencing.
4.
Matching the signature to the genome.
Each of the sequenced
17-mer signatures (which typically matches only one position
in a complex genome [7]) is associated with a proximal gene.
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