Applied Biosystems SOLiD™ 4 System SETS Software User Guide ...

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Chapter 6 Support Multiplexing
Fragment and Paired-End Multiplexing
Fragment and Paired-End Multiplexing
Overview of
Multiplexing and
Barcodes
Multiplexing in general, and barcodes in particular, enable the
SOLiD 4 System to target many applications, from deep
sequencing of a single source to extreme parallel sequencing of a
large number of samples over limited regions - sometimes on the
same slide.
The additional stretches of DNA sequence, known as barcodes, can
be used to segregate pooled library samples during sequencing back
into their original (logical) single library groupings. Barcodes –in
their simplest form– allow the pooling of samples on a slide without
the loss of surface area creating geographic isolations. Multiplexing
occurs at both the whole slide level as well as at the geographically
isolated (mask) region level.
A barcode, by definition, is a tag with a known sequence that is
attached to a sequencing primer on a bead during library preparation.
When sequenced, it is the barcode that identifies and classifies the
bead to a named sample. This barcoding, or indexing, approach
allows multiplexing at the sequencing level.
Following sequencing of the target DNA, additional rounds of
ligation-based sequencing are performed using primer sets
complimentary to the barcode. The resulting reads can then be sorted
by the barcode and aligned in groups to the reference sequence.
Barcodes are assigned to a library both conceptually and physically
(as specific tags) as part of the library preparation. If more than one
barcode is assigned to a single library during the library preparation
step, the conceptual grouping is referred to as a barcode group. All
barcodes in the pool will assign the underlying beads to the same
original library for analysis.
The SOLiD 4 System supports multiplexing for both fragment
sequencing and paired-end sequencing. See “Naming convention for
sequencing reads” on page 11.
Note: Multiplexing is not available for mate-paired libraries.
104 Applied Biosystems SOLiD 4 System SETS Software User Guide