Editing BioMicroCenter:Element Sequencing (section)

===Requirements/Things to Consider===
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<LI>Polony-mapping-generation (PMG), is done on the first 5 reads of read one.  You will want to have AT LEAST these 5 positions with high diversity (roughly equal A,G,C, and T's across the flow cell per read).  However, the AVITI24 does give us the option to PMG off index 1 (sp27), with a suggested multiplex of 64 or more.  PMG can also be done upstream of the start of read 1, up to a total of 20 bases.  However, these bases will be read as dark.  You will not received data on these bases, and you will lose those cycles.  If these options can not be applied, then we will need to increase the percent phiX control spiked into the final loading sample by as much as 30% to increase the read 1 diversity.
<LI>Illumina-ready-linear libraries (have p5/p7 flow cell binding sites) are preferred as these tend sequence much more efficiently on the Freestyle flow cell, and can be applied to other platforms should the need arise. 
<LI>Element-ready-linear libraries (sp5/sp27 flow cell binding sites) must have a 5'-phosphate on the sp5-end sequence in order to circularize on the AVITI.  If you choose to use Element's flow cell binding sites, sp5 and sp27, it's suggested that library prep primers are HPLC cleaned and used within a month or so as any truncated primers, as part of the primer creation process or due to degradation over time, are unlikely to circularize leading to a decrease in read count.
<LI>Library preparations involving biotin-modifications, poly-A tailing, and/or bead-based normalization will not sequence well, if at all.
<LI>Read direction will be just like MiSeq.  The index reads are sequenced first, starting with the sp27 index (which will be read off the sequencing primer site), followed by the SP5 index (which will be read off the sp5 flow cell binding sequence).  The read sequences are then read starting with read 1 (using the Read 1 primer sequence), followed by the amplification of the of the reverse strand.  Read 2 is then read using the reverse complement of the Read 2 primer sequence.