To validate calls for SNP variation between the reference genome and the sequenced strains, the corresponding regions were PCR amplified and Sanger sequenced (Big Dye Terminator V1.1 Cycle sequencing kit from Life Technologies) using standard conditions.
The resulting deletion cassette was verified by PCR and nucleotide sequencing (Big Dye kit, ABI Perking Elmer).
Sequencing of the amplified products was done on an automated Applied Biosystems' 3130 XL system using cycle sequencing big dye terminator (ABI).
PCR-fragments of 1,809 (HA gene) and 1,362 nucleotides (NA gene) were obtained and sequenced (Big Dye Terminator v1.1 Cycle Sequencing kit, Applied Biosystems, Foster City, CA,USA).
DNA sequences were performed on both strands using automatic sequencing (Big Dye terminator chemistry, Applied Biosystems, Foster City, CA, USA).
Restriction positive colonies were DNA sequenced (Big Dye® Terminator Cycle Sequencing Kit, Applied Biosystems) using a primer located at the 3' end of the U6 promoter (5' CACAAAAGGA AACTCACCC 3').
The resulting deletion cassettes slr0946::Kmr and slr1738 Kmr were sequenced (Big Dye kit, ABI Perking Elmer) prior to transformation to Synechocystis.
All different genotypes found in the PSQ reaction were confirmed by sequencing (Big Dye, Terminator Cycle Sequencing; Applied Biosystems) according to the manufacturer's instructions and run on an ABI 3100 Genetic Analyzer (Applied Biosystems).
After purification (Microcon-PCR Filter Unit, Millipore, Paris, France), the PCR products were sequenced (Big Dye Terminator v1.1 Cycle sequencing kit, Applied Biosystems, Forster City, CA, USA) and analysed on ABI PRISM 3100 Genetic Analyser (Applied Biosystems).
PCR products for GHR and CYB were purified from 1% agarose gels using Amicon Ultrafree-DNA columns (Millipore) and sequenced on both strands using automatic sequencing (Big Dye Terminator cycle kit) on an ABI 310 (PE Applied Biosystems).
