Computational protocol: High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

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Protocol publication

[…] Following Sanger sequencing, hdc reads were analysed using the NCBI nucleotide database (BlastN; http://blast.ncbi.nlm.nih.gov/). Sanger sequencing of the tdc amplicons did not provide forward and reverse reads of the complete 1100 bp, therefore, only the overlap (approximately 800 bp), aligned using the MegAlign programme was analysed using the BlastN database.Raw Ion PGM reads were quality filtered with the fastq_filter script in USEARCH. For both tdc and hdc amplicons, a length cut-off of 170 bp was used. Reads were then clustered into operational taxonomical units (OTUs) and chimeras removed with the 64-bit version of USEARCH []. Subsequently OTUs were aligned with MUSCLE [] and a phylogenetic tree generated within Qiime []. Alpha diversity analysis was also implemented within Qiime. For taxonomic assignment OTUs were blasted against the NCBI-Nr database and parsed through MEGAN []. […]

Pipeline specifications

Software tools BLASTN, USEARCH, MUSCLE, QIIME, MEGAN
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Lactobacillus curvatus, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus
Chemicals Amines, Histamine, Tyramine, Tyrosine