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3.3.2.2. Reduced cycle amplification:
The next step is called reduced cycle amplification. During this step, sequences for
primer binding, indices, and terminal sequences are added. Indices are usually six base
pairs long and are used during DNA sequence analysis to identify samples. Indices allow
for up to 96 different samples to be run together. During analysis, the computer will
group all reads with the same index together. The terminal sequences are used for
attaching the DNA strand to the flow cell. Illumina uses a "sequence by
synthesis" approach. This process takes place inside of an acrylamide-coated glass
flow cell. The flow cell has oligonucleotides (short nucleotide sequences) coating the
bottom of the cell, and they serve to hold the DNA strands in place during sequencing.
The oligos match the two kinds of terminal sequences added to the DNA during reduced
cycle amplification. As the DNA enters the flow cell, one of the adapters attaches to a
complementary oligo.
3.3.2.3. Bridge amplification:
Once attached, cluster generation can begin. The goal is to create hundreds of identical
strands of DNA. Some will be the forward strand; the rest, the reverse. Clusters are
generated through bridge amplification. Polymerases move along a strand of DNA,
creating its complementary strand. The original strand is washed away, leaving only the
reverse strand. At the top of the reverse strand there is an adapter sequence. The DNA
strand bends and attaches to the oligo that is complementary to the top adapter sequence.
Polymerases attach to the reverse strand, and its complementary strand (which is
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identical to the original) is made. The now double stranded DNA is denatured so that
each strand can separately attach to an oligonucleotide sequence anchored to the flow
cell. One will be the reverse strand; the other, the forward. This process is called bridge
amplification, and it happens for thousands of clusters all over the flow cell at once.
3.3.2.4. Clonal amplification
Over and over again, DNA strands will bend and attach to oligos. Polymerases will
synthesize a new strand to create a double stranded segment, and that will be denatured
so that all of the DNA strands in one area are from a single source (clonal amplification).
Clonal amplification is important for quality control purposes. If a strand is found to
have an odd sequence, then scientists can check the reverse strand to make sure that it
has the complement of the same oddity. The forward and reverse strands act as checks to
guard against artifacts. Because Illumina sequencing uses polymerases, base substitution
errors have been observed, especially at the 3' end. Paired end reads combined
with cluster generation can confirm an error took place. The reverse and forward strands
should be complementary to each other, all reverse reads should match each other, and
all forward reads should match each other. If a read is not similar enough to its
counterparts (with which it should be a clone), an error may have occurred. A minimum
threshold of 97% similarity has been used in some labs' analyses
3.3.2.5. Sequence by synthesis