To have the luxury of a little separate work space is huge — and to have the dream-sequence master bath".
Sequencing was performed following PCR amplification in a 10 μl reaction containing 50 ng template DNA, 0.75× sequencing buffer, 500 nM primers and 1 ul BigDye v1.1 sequencing master mix (Applied Biosystems) with an initial denaturation at 98°C for 2 minutes, followed by 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds and 60°C for 4 minutes.
To ensure sufficient volume for instrument pipetting, sequencing master mixes are prepared in 15 mL conical tubes using 6958 μL deionized water, 5219 μL dilution buffer, and 1740 μL BigDye® v1.1 (for forward sequencing; 1281 μL BigDye® v1.1 plus 427 μL dGTP BigDye® v1.1 is used instead for reverse sequencing).
As a result, at high mutation rates these sequences compete with the master sequence (the single sequence that has higher fitness than all other sequences), and can win this competition at sufficiently high mutation rate by sheer abundance.
As the mutant phenotype is composed of all the mutant sequences except the master sequence, back mutation can be neglected as a first approximation.
The classic approach of Benders decomposition algorithm is implemented, and it solves an alternative sequence of master and subproblems.
Users can then decide to mark a specific sequence as master.
The sequencing revealed only one master sequence with a few point mutations (Supplementary Figure S1C).
The focus in the present study is on cases which exhibit only a single compotype each (NC = 1), whereby compositional entities are disposed around a single center of mass, analogous to the master sequence in sequence-based quasispecies.
In both cases, the quasispecies have no persisting master sequence, and the sequence variability in the field is high.
In particular, we propose a novel definition of chromosomal master sequence, as a sequence of physically distinct whole or fragmented chromosomes, whose length is taken to be the sum of the copy numbers of each whole or fragmented chromosome.
