Illumina MiSeq sequencing
Replicate 3
Illumina sequencing was performed on amplicons obtained with primers on the 24 genomic DNA samples from sedge warblers. Sequencing primers were designed including universal Illumina 5’ tails followed by unique 6bp barcode sequences and the specific primers designed to amplify 235-241bp fragments of exon 3, encoding the α2 domain of the antigen-binding groove of the MHC class I sedge warbler molecule (forward: GAGYGGGGGTCTCCACAC, reverse: TGCGMTCCAGYTCCTTCTGCCC). The specific primers were designed based on cDNA and gDNA sequences obtained from warblers (Biedrzycka, Radwan and Westerdahl, unpublished). Amplification was performed with HotStar Master Mix (Qiagen) with the following parameters: 95oC 30 s, 27 cycles of 66oC 30 s, 72oC 90 s. PCR products were pooled in equimolar quantities and purified (MinElute PCR Purification Kit - Qiagen). Pooled amplicons were amplified in order to add Illumina P5/P7 adaptor sequences (Syed et al. 2009). The pool was diluted from 10x to 100x, depending on the density of the electrophoresis band, and PCR utilizing primers carrying P5/5 adaptors was performed for 12 cycles (according to Illumina MiSeq protocol). Obtained products were purified with the MinElute PCR Purification Kit (Qiagen) and paired-end sequencing was performed on Illumina Miseq with the Miseq Reagent Kit v2 for 300 cycles (Illumina, Inc., San Diego, CA, USA). Two replicates of the 24 samples, amplified in two independent PCRs and run in two separate sequencing experiments (runs 1 and 2) were amplified using three forward and eight unique reverse tags, such that each individual could be recognised by a unique combination. These two replicates were used to calculate reliability of genotyping within and between methods. The same 24 individuals were amplified a third time with 24 forward and 24 reverse tags (run 3), such that none of the tags was shared among individuals. The aim of this third replication was to assess the magnitude of “tag jumping” between individuals during the second PCR where pooled amplicons are amplified in one PCR reaction (Schnell et al. 2015), i.e. the potential forming of chimeras between sequences from different individuals that would results in an exchange of 3’ tags.  Primers and tags used in AmpliSAS format (Sebastian et al. 2015):  Runs 1 and 2: >marker,length,primer_f,primer_r,gene,feature,specie MHCI,235 238 241,GAGYGGGGGTCTCCACAC,TGCGMTCCAGYTCCTTCTGCCC,MHCI,ex3,Acrocephalus schoenobaenus >sample,tag_f,tag_r 37,ACAACC,AGCCTC 38,ACAACC,TCGTTA 39,ACAACC,TGTGGC 40,ACAACC,CTCTGC 41,ACAACC,CCTAAT 42,ACAACC,ATGGAT 43,ACAACC,ACAGGT 44,ACAACC,TCCGCT 45,TCAGAG,AGCCTC 46,TCAGAG,TCGTTA 47,TCAGAG,TGTGGC 48,TCAGAG,CTCTGC 49,TCAGAG,CCTAAT 50,TCAGAG,ATGGAT 51,TCAGAG,ACAGGT 52,TCAGAG,TCCGCT 53,GTAGTG,AGCCTC 54,GTAGTG,TCGTTA 55,GTAGTG,TGTGGC 56,GTAGTG,CTCTGC 57,GTAGTG,CCTAAT 58,GTAGTG,ATGGAT 59,GTAGTG,ACAGGT 60,GTAGTG,TCCGCT  Run 3: >marker,length,primer_f,primer_r,gene,feature,specie MHCI,235 238 241,GAGYGGGGGTCTCCACAC,TGCGMTCCAGYTCCTTCTGCCC,MHCI,ex3,Acrocephalus schoenobaenus >sample,tag_f,tag_r 37,GGTTCT,AACCGA 38,GTAACA,CCGGAA 39,AATCCT,AGTGTT 40,AGACCG,CCGCTG 41,CTATAA,AACGCG 42,AATGAA,GGCTAC 43,GTACAC,TTCTCG 44,TCGACG,TCACTC 45,TTCGGA,AGCCTC 46,TGTACA,TCGTTA 47,CTCATG,TGTGGC 48,CATACC,CTCTGC 49,ACAACC,CCTAAT 51,GTAGTG,ACAGGT 52,AGCACT,TCCGCT 54,ACACAA,GAAGCT 55,GCTCCG,GATATT 56,TACTTC,CATTGA 57,GTCTTA,AGCTGG 58,GTATGT,CGCGAT 59,TGCCAG,ACATTG 60,GTCAAT,CCAAGG
ADAM MICKIEWICZ UNIVERSITY, POZNAN, POLAND
ena-EXPERIMENT-ADAM MICKIEWICZ UNIVERSITY, POZNAN, POLAND-13-11-2015-14:52:20:571-3