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1.
PLoS Comput Biol ; 13(5): e1005480, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28475621

RESUMEN

Unique molecular identifiers (UMIs) show outstanding performance in targeted high-throughput resequencing, being the most promising approach for the accurate identification of rare variants in complex DNA samples. This approach has application in multiple areas, including cancer diagnostics, thus demanding dedicated software and algorithms. Here we introduce MAGERI, a computational pipeline that efficiently handles all caveats of UMI-based analysis to obtain high-fidelity mutation profiles and call ultra-rare variants. Using an extensive set of benchmark datasets including gold-standard biological samples with known variant frequencies, cell-free DNA from tumor patient blood samples and publicly available UMI-encoded datasets we demonstrate that our method is both robust and efficient in calling rare variants. The versatility of our software is supported by accurate results obtained for both tumor DNA and viral RNA samples in datasets prepared using three different UMI-based protocols.


Asunto(s)
Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Programas Informáticos , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Bases de Datos Genéticas , Humanos , Neoplasias/genética , ARN Viral/genética , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ARN/métodos
2.
J Immunol ; 194(12): 6155-63, 2015 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-25957172

RESUMEN

Emerging high-throughput sequencing methods for the analyses of complex structure of TCR and BCR repertoires give a powerful impulse to adaptive immunity studies. However, there are still essential technical obstacles for performing a truly quantitative analysis. Specifically, it remains challenging to obtain comprehensive information on the clonal composition of small lymphocyte populations, such as Ag-specific, functional, or tissue-resident cell subsets isolated by sorting, microdissection, or fine needle aspirates. In this study, we report a robust approach based on unique molecular identifiers that allows profiling Ag receptors for several hundred to thousand lymphocytes while preserving qualitative and quantitative information on clonal composition of the sample. We also describe several general features regarding the data analysis with unique molecular identifiers that are critical for accurate counting of starting molecules in high-throughput sequencing applications.


Asunto(s)
Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Recuento de Linfocitos , Linfocitos/metabolismo , Biología Computacional/métodos , ADN Complementario , Perfilación de la Expresión Génica/métodos , Humanos , Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Posición Específica de Matrices de Puntuación , Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética
3.
PLoS One ; 12(9): e0184507, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28892497

RESUMEN

Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.


Asunto(s)
Cartilla de ADN , Dosificación de Gen , Genoma Humano , Genómica , Reacción en Cadena de la Polimerasa/métodos , Variaciones en el Número de Copia de ADN , Biblioteca de Genes , Genómica/métodos , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos
4.
Protein Sci ; 12(9): 2001-14, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12930999

RESUMEN

Fast and accurate side-chain conformation prediction is important for homology modeling, ab initio protein structure prediction, and protein design applications. Many methods have been presented, although only a few computer programs are publicly available. The SCWRL program is one such method and is widely used because of its speed, accuracy, and ease of use. A new algorithm for SCWRL is presented that uses results from graph theory to solve the combinatorial problem encountered in the side-chain prediction problem. In this method, side chains are represented as vertices in an undirected graph. Any two residues that have rotamers with nonzero interaction energies are considered to have an edge in the graph. The resulting graph can be partitioned into connected subgraphs with no edges between them. These subgraphs can in turn be broken into biconnected components, which are graphs that cannot be disconnected by removal of a single vertex. The combinatorial problem is reduced to finding the minimum energy of these small biconnected components and combining the results to identify the global minimum energy conformation. This algorithm is able to complete predictions on a set of 180 proteins with 34342 side chains in <7 min of computer time. The total chi(1) and chi(1 + 2) dihedral angle accuracies are 82.6% and 73.7% using a simple energy function based on the backbone-dependent rotamer library and a linear repulsive steric energy. The new algorithm will allow for use of SCWRL in more demanding applications such as sequence design and ab initio structure prediction, as well addition of a more complex energy function and conformational flexibility, leading to increased accuracy.


Asunto(s)
Proteínas/química , Proteómica/métodos , Algoritmos , Biología Computacional , Simulación por Computador , Bases de Datos como Asunto , Disulfuros , Modelos Moleculares , Modelos Estadísticos , Conformación Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Programas Informáticos
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