bacteriome of adelie penguin guano sampled in march
Pooled samples from each month were examined. Phylogenetic study was performed 149 by sequencing and analysis of prokaryotic 16S ribosomal RNA gene. A fragment of the16S 150 rRNA gene containing the V3 and V4 variable regions was amplified using gene-specific 151 primers: 16S_V3-F and 16S_V4-R positions 341-357F and 785-805R, respectively, according 152 to Escherichia coli 16S rRNA gene reference sequence (Klindworth et al. 2013). Illumina 153 Nextera XT overhang adapter nucleotide sequences were included in addition to the 16S 154 rRNA gene-specific sequences, which allowed sample indexing and pooling. Each PCR 155 amplification was done in triplicate using KAPA HiFiPCR in a final volume of 20 L per 156 reaction according to the manufacturers instructions. Obtained PCR products were pooled in 157 equimolar ratio and indexed using Nextera XT barcodes (Illumina, San Diego, USA). 158 Amplicon libraries were pooled and sequenced on Illumina MiSeq instrument (Illumina, San 159 Diego, USA) in the DNA Sequencing and Oligonucleotide Synthesis Laboratory (Institute of 160 Biochemistry and Biophysics, Polish Academy of Sciences). Sequencing was done in paired-161 end mode (2  300 bp) with the use of a v3 (600 cycles) chemistry cartridge which allowed 162 generation of long paired reads fully covering 16S V3V4 amplicons.
MG1