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 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
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Integrations