Page Comparison

NTUni_p5 and NTUni_p7 (these are the Nextera transposes sequences, so their matching sequencing primers will be in the Illumina sequencing kits):
  TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
                |||||||||||||||||||
 CAGAGCACCCGAGCCTCTACACATATTCTCTGTC
(so this can't be an A-tailed - the extra "A" isn't in the sequencing primer and would cause base diversity issues during sequencing)

Now add the metagenoimcs primers (Hyb_F01_i5 with "ATCACG" and Hyb_R21_i7 with "CGAAAC" (rev comp) in this example) which get incorporated AFTER CYCLE 1:


Cycle 1 - only Hyb_R21_i7 hybridizes and extends to form the reverse compliment of the top strand:
                                     TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG <insert> CTGTCTCTTATACACATCTCCGAGCCCACGAGAC
                                                   ||||||||||||||||||| <insert> |||||||||||||||||||
                                    CAGAGCACCCGAGCCTCTACACATATTCTCTGTC <insert> GACAGAGAATATGTGTAGACTGCGACGGCTGCT
                                                                                                   GGCTCGGGTGCTCTG CAAAGC TAGAGCATACGGCAGAAGACGAAC <- reverse primer binds in cycle 1
 
Cycle 2 - the newly-formed top strand now has the compliment to the forward primer which can now prime:
AATGATACGGCGACCACCGAGATCTACAC ATCACG TCGTCGGCAGCGTC <- forward primer can now bind
                                     TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG <insert> CTGTCTCTTATACACATCTCCGAGCCCACGAGAC
                                                   ||||||||||||||||||| <insert> |||||||||||||||||||
                                     AGCAGCCGTCGCAGTCTACACATATTCTCTGTC <insert> GACAGAGAATATGTGTAGACTGCGACGGCTGCTG CAAAGC TAGAGCATACGGCAGAAGACGAAC <- made in cycle 1
                                                                                                   GGCTCGGGTGCTCTG CAAAGC TAGAGCATACGGCAGAAGACGAAC <- reverse primer

this is all happening upside-down to the other strand in cycle 1