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Sequencing

AK Alexander Kula
JS Joseph Saelens
JC Jennifer Cox
AS Alyxandria M. Schubert
MT Michael Travisano
CP Catherine Putonti
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Genomic extraction was performed of a single genotype per collection time using the UltraCleanTM Microbial DNA Isolation kit as described previously. Twelve primer pairs were designed using the Primer3 web-application72; when all twelve pairs are used, a minimum 2 × coverage of the genome is possible (primer sequences available upon request). PCR products were purified using ExoSAP-It and sequenced by the University of Chicago Cancer Research Center DNA Sequencing Facility.

Sequencing of the complete genome with a 4 × coverage was conducted after the 1st, 5th, 11th, 21st, and 35th transfers for the S and E lines and after the 50th transfer for the J lines. The C1 line was also sequenced after the1st 5th, 11th, 21st and 35th transfers. Sequences of the final evolved lines for the C1 line (GenBank: HM775306), S line (GenBank: HM775307), E1 line (GenBank: HM775308), E2 line (GenBank: HM775309), E3 line (GenBank: HM775310), and E4 line (GenBank: HM775311). The three J lines have been deposited as well (GenBank numbers being processed).

Sequencing at each collection time was conducted initially by extracting viral DNA from lysate. As such, the potential for numerous genotypes to be pooled existed. Additionally, we also plated via serial dilutions lysate collected and selected plaques at random for sequencing. In all cases the same genotype was recovered suggesting relatively low heterogeneity within the population.

Copyright and License information: The Author(s) ©2018
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