Computational protocol: Genome-Wide Sequence Variation among Mycobacterium avium Subspecies paratuberculosis Isolates: A Better Understanding of Johne’s Disease Transmission Dynamics

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Protocol publication

[…] Raw sequences obtained from the Illumina GAIIx were analyzed using the CLC Genomic Workbench software (version 4.0.3, CLC Bio, Cambridge, MA, USA) to perform both de novo and comparative reference assembly. For the M. ap genomes, all sequences were assembled in reference to the revised M. ap K-10 sequences (Wynne et al., ). The M. avium DT 78 genome was assembled using the genome of M. avium subsp. hominissuis 104 (NCBI accession NC 008595) as a reference. The de novo assembly was used for the genome sequence of Env 77 strain because of the lack of significant similarity to other genomes. Additionally, the MAUVE algorithm was used to align paired or multiple genomes for comparative purposes, as outlined before (Perna et al., ; Darling et al., ). The gapped consensus sequence of each strain was imported to MAUVE for sequence alignment at default seed weight setting. [...] For SNPs detection, we used algorithms implemented in CLC Bio Workstation. Criteria for identifying SNPs included a coverage range setting at 10–55 reads and a presence frequency in at least 50% of the reads before consideration for further analysis. A randomly selected number of SNPs were further analyzed using Sanger sequencing to confirm Next-Generation sequencing data. The primers were designed to cover 10 possible SNPs. The BigDye Terminator (Applied Biosystems, Foster City, CA, USA) version 3.1 cycle sequencing kit was used for sequencing. The sequencing PCR included an initial denaturation cycle at 95°C for 5 min followed by 35 cycles of 95°C for 20 s, 45°C for 30 s and 60°C for 2 min with a final extension at 72°C for 7 min. All samples were sent to the Biotechnology Center at the University of Wisconsin–Madison for sequencing on a ABI 3730XL machine (Applied Biosystems).For the genome-wide phylogeny (phylo-genome analysis), the predicted SNPs from sequenced genomes (M. ap isolates) and the corresponding nucleotides in DT 78, M. ap K-10 and M. avium 104 were tabulated to create a concatenated sequences of each strain. The genome of M. avium Env 77 isolate was excluded from such analysis because of the low similarity to other genomes. The concatenated sequence of each strain was aligned using CLUSTALW, and phylogenetic trees were generated with MEGA version 5 using one of the following methods: maximum parsimony (MP), maximum likelihood (ML), maximum likelihood with molecular clock (MLK) assumption in addition to Neighbor-joining algorithm with a bootstrapping values of 1,000 replicates applied to all methods (Tamura et al., ). […]

Pipeline specifications

Software tools Mauve, Clustal W, MEGA
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Mycobacterium avium, Homo sapiens, Bos taurus
Diseases Infection, Paratuberculosis