RESUMO
Sanger sequencing is a common method of reading DNA sequences. It is less expensive than high-throughput methods, and it is appropriate for numerous applications including molecular diagnostics. However, sequencing mixtures of similar DNA of pathogens with this method is challenging. This is important because most clinical samples contain such mixtures, rather than pure single strains. The traditional solution is to sequence selected clones of PCR products, a complicated, time-consuming, and expensive procedure. Here, we propose the base-calling with vocabulary (BCV) method that computationally deciphers Sanger chromatograms obtained from mixed DNA samples. The inputs to the BCV algorithm are a chromatogram and a dictionary of sequences that are similar to those we expect to obtain. We apply the base-calling function on a test dataset of chromatograms without ambiguous positions, as well as one with 3-14% sequence degeneracy. Furthermore, we use BCV to assemble a consensus sequence for an HIV genome fragment in a sample containing a mixture of viral DNA variants and to determine the positions of the indels. Finally, we detect drug-resistant Mycobacterium tuberculosis strains carrying frameshift mutations mixed with wild-type bacteria in the pncA gene, and roughly characterize bacterial communities in clinical samples by direct 16S rRNA sequencing.
Assuntos
Algoritmos , Biologia Computacional/métodos , Análise de Sequência de DNA , Genótipo , HIV-1/genética , Vírus de Hepatite/classificação , Vírus de Hepatite/genética , Humanos , Mutação INDEL , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Filogenia , RNA Ribossômico 16SRESUMO
The molecular basis of potassium uptake in cyanobacteria has not been elucidated. However, genes known from other bacteria to encode potassium transporters can be identified in the genome of Synechocystis sp. strain PCC 6803. Mutants defective in kdpA and ntpJ were generated and characterized to address the role of the Kdp and KtrAB systems in this strain. KtrAB is crucial for K(+) uptake, as the DeltantpJ mutant shows slowed growth, slowed potassium uptake kinetics, and increased salt sensitivity. The DeltakdpA mutant has the same phenotype as the wild type even at limiting potassium, but a DeltakdpADeltantpJ double mutant is not viable, indicating a role of Kdp for potassium uptake when the Ktr system is not functioning.
Assuntos
Proteínas de Transporte de Cátions/fisiologia , Cianobactérias/metabolismo , Proteínas de Membrana/fisiologia , Potássio/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Proteínas de Transporte de Cátions/genética , Cianobactérias/química , Cinética , Proteínas de Membrana/genética , Mutação , Fenótipo , Sais/farmacologiaRESUMO
A collection of 17 salt-sensitive mutants of the cyanobacterium Synechocystis sp. strain PCC 6803 was obtained by random cartridge mutagenesis. The genes coding for proteins essential for growth at high salt concentrations were mapped on the completely known genome sequence of this strain. The two genes coding for enzymes involved in biosynthesis of the osmolyte glucosylglycerol were affected in nine mutants. Two mutants defective in a glycoprotease encoding gene gcp showed a reduced salt resistance. Four genes were identified not previously known to be essential for salt tolerance in cyanobacteria. These genes (slr1799, slr1087, sll1061, and sll1062) code for proteins not yet functionally characterized.
