Ferla Matteo Paolo
Formerly Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Present address: Biosyntia, DTU Centre for Biosustainability, Hørsholm, Denmark.
BMC Bioinformatics. 2016 Apr 4;17:152. doi: 10.1186/s12859-016-0996-7.
Assessing library diversity is an important control step in a directed evolution experiment. To do this, a limited amount of colonies from a test library are sequenced and tested. In the case of an error-prone PCR library, the spectrum of the identified mutations - the proportions of mutations of a specific nucleobase to another- is calculated enabling the user to make more informed predictions on library diversity and coverage. However, the calculations of the mutational spectrum are severely affected by the limited sample sizes.
Here an online program, called Mutanalyst, is presented, which not only automates the calculations, but also estimates errors involved. Specifically, the errors are calculated thanks to the complementarity of DNA, which means that a mutation has a complementary mutation on the other sequence. Additionally, in the case of determining the mean number of mutations per sequence it does so by fitting to a Poisson distribution, which is more robust than calculating the average in light of the small sampling size.
As a result of the added measures to keep into account of small sample size the user can better assess whether the library is satisfactory or whether error-prone PCR conditions should be adjusted. The program is available at www.mutanalyst.com .
评估文库多样性是定向进化实验中的一个重要控制步骤。为此，对测试文库中的少量菌落进行测序和测试。对于易错PCR文库，计算所鉴定突变的频谱——特定核碱基与另一个核碱基的突变比例——可让用户对文库多样性和覆盖率做出更明智的预测。然而，突变频谱的计算受到样本量有限的严重影响。
本文介绍了一个名为Mutanalyst的在线程序，它不仅能自动进行计算，还能估计其中涉及的误差。具体而言，由于DNA的互补性计算出误差，这意味着一个突变在另一条序列上有一个互补突变。此外，在确定每条序列的平均突变数时，它通过拟合泊松分布来进行，这比根据小样本量计算平均值更可靠。
由于采取了考虑小样本量的附加措施，用户可以更好地评估文库是否令人满意，或者是否应调整易错PCR条件。该程序可在www.mutanalyst.com上获取。
