Editing BioMicroCenter:Singular Sequencing (section)

===Requirements/Things to Consider===
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<LI>All 4 bases (A, G, C, T) must appear within the first 10 cycles of read 1.
<LI>Both forward and reverse read strands are present and accessible during sequencing.  After the desired number of cycles have been reached for a given read, a block is introduced to prevent any continued extension of the growing read chain, but the generated read chain is NOT denatured/melted away before the priming and extension of the next read.  For this reason, lane-by-lane requests must be either 50 or 150 paired-end compatible with a dual index of 8 nt each.  All other requests will require a 4 lane (or 1 flow cell) minimum.
<LI>Indexes are read off flow cell binding regions, S1 and S2.  This is unlike modern Illumina platforms, which read off of sequencing priming regions of the final library (SP1 and SP2 in diagram above).  For standard lane-by-lane requests, the first index will be read off the S1 region of the final library, and index 2 will be read off the S2 region.  Compared to a typical Illumina library, a Singular index 1 would be the reverse compliment of the i5 index, whereas index 2 would be the reverse compliment of the i7 index.
<LI>Each flow cell runs off a single reagent cartridge, thus each lane on that flow cell will be treated with the same primer cocktail by run.  For standard lane-by-lane requests, this means all custom primers must be compatible with TruSeq, Nextera, TruSeq smaRNA, and Solexa sequencing primers.
<LI>Singular also supports the conversion of Illumina libraries through either index replacement or anchor expansion for completed libraries that wish to be run on this platform.  Index hopping rates are near zero, limiting the need for UDI's.  Early quality metrics can be seen below.