48 results about "Massive parallel sequencing" patented technology

Disclosed is a method to achieve digital quantification of DNA (i.e., counting differences between identical sequences) using direct shotgun sequencing followed by mapping to the chromosome of origin and enumeration of fragments per chromosome. The preferred method uses massively parallel sequencing, which can produce tens of millions of short sequence tags in a single run and enabling a sampling that can be statistically evaluated. By counting the number of sequence tags mapped to a predefined window in each chromosome, the over- or under-representation of any chromosome in maternal plasma DNA contributed by an aneuploid fetus can be detected. This method does not require the differentiation of fetal versus maternal DNA. The median count of autosomal values is used as a normalization constant to account for differences in total number of sequence tags is used for comparison between samples and between chromosomes.

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called “Safe-SeqS” for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant (“super-mutants”) if ≧95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

Embodiments are provided that provide for parallel sequencing of nucleic acid segments. In some embodiments, a single sequence is sequenced by at least two different sequencing techniques and the results compared, allowing for deficiencies or strengths of one technique to be complemented by the second technique.

Massively parallel sequencing of cell-free, maternal plasma DNA was recently demonstrated to be a safe and effective screening method for fetal chromosomal aneuploidies. Here, we report an improved sequencing method achieving significantly increased throughput and decreased cost by replacing laborious sequencing library preparation steps with PCR employing a single primer pair. Using this approach, samples containing as little as 4% trisomy 21 DNA could be readily distinguished from euploid samples.

Described herein is a buffer concentration for highly efficient ligation of two oligonucleotides. The embodiments herein have led to the development of an optimized ligation step used in the sample preparation for sequencing reactions. Further, embodiments herein describe a high-throughput method for sequencing using barcodes or the purpose of multiplexing several samples simultaneously and novel methods for making targeted DNA libraries for re-sequencing on massively parallel next-generation sequencing platforms and for alternatives to gel-purification for recovering the desired templates from small RNA libraries for next generation sequencing.

An establishment method of a gene library of fecal flora based on high-throughput gene sequencing employs a ''nested PCR'' method for enrichment amplification on 16S rDNA, so as to reduce the host and food residue genome pollution by the maximum; and V3 and V6 are combined for specific amplification and massive parallel sequencing, so as to obtain the a target gene sequence database of the flora. The library can increase the identification of the bacterial flora from Genus level to Species level. The method can maximally avoid the interference of host cell nucleic acid, and completely and efficiently detect 16SrDNA tag sequence of all bacteria varieties, so as to accurately determine the ecological structure of the intestinal flora.

The present invention provides methods, kits and compositions for enriching target sequences from a sequencing library to provide a target enriched sequencing library, wherein the sequencing library is suitable for massive parallel sequencing and comprises a plurality of double-stranded nucleic acid molecules.

The invention provides methods for non-invasive prenatal testing that allow for detecting risk of chromosomal and subchromosomal abnormalities, including but not limited to aneuploidies, microdeletions and microduplications, insertions, translocations, inversions and small-size mutations including point mutations and mutational signatures. The methods of the invention utilize a pool of TArget Capture Sequences (TACS) to enrich for sequences of interest in a mixed sample containing both maternal and fetal DNA, followed by massive parallel sequencing and statistical analysis of the enriched population to thereby detect the risk of a genetic abnormality in the fetal DNA. Kits for carrying out the methods of the invention are also provided.

This invention relates to a method and apparatus for image processing, and more particularly, this invention relates to a method and apparatus for processing image data generated by bioanalytical devices, such as DNA sequencers. An object of the present invention is to remove artifacts such as noise, blur, background, non-uniform illumination, lack of registration, and extract pixel signals back to DNA-beads in a way that de-mixes pixels that contain contributions from nearby beads. In one aspect of the present invention, a system for optimizing an image comprises means for receiving an initial image which includes a plurality of microparticles with different intensities; a computing device, comprising a processor executing instructions to perform: generating an initial function denoting each microparticle's location and intensity in the initial image; determining an image processing operator adapted to determine an extent of point spread and blurriness in the initial image; computing an optimum function denoting each microparticle's location and intensity in an optimizing image; and producing the optimizing image with enhanced accuracy and density of the microparticles.

The invention discloses 29 micro-haplotype sites, a screening method, a multiplex amplification system and an application. The screening method comprises the following steps: acquiring population data, acquiring micro-haplotype genotypes and allele frequencies from the population data, calculating forensic related parameters of candidate sites in different populations and excluding sites affectingsubsequent sequencing typing to obtain the 29 micro-haplotype sites; designing primers and amplification conditions according to the 29 micro-haplotype sites, and establishing the multiplex amplification system based on three-round PCR amplification systems. According to the multiplex amplification system, DNA templates extracted from mixed biological samples are amplified, amplification resultsare subjected to large-scale parallel sequencing, value of the sequencing depth of each micro-haplotype site in the DNA templates is obtained, and finally, the purpose of typing the mixed biological samples is achieved. The multiplex amplification system is suitable for large-scale parallel sequencing technology and has the advantages of high detection flux, high detection speed, large quantity ofobtained data, more accurate detection results and the like.

The present invention provides a method for the isolation and analysis of a target nucleic acid, the target nucleic acid being present in a sample of genomic DNA, comprising the steps of a) fragmentation of the genomic DNA, b) hybridization of the genomic DNA on a nucleic acid solid support, the solid support comprising a plurality of oligonucleotide probes, the probes being characterized in that each probe is at least partially complementary to the sequence of the target nucleic acid or its complement, under hybridization conditions, characterized in that the plurality of probes hybridizes to fragments of the target nucleic acid but does not hybridize to other nucleic acids which are present in the sample, c) stripping off the target molecules hybridized to the nucleic acid array, d) overlap extension synthesis in order to generate double stranded overlap extension synthesis product, e) fragment polishing, and f) adaptor ligation.

There is disclosed a method of generating a massively parallel sequencing library comprising the steps of: a) providing a primary WGA DNA library (pWGAlib), including fragments comprising a WGA library universal sequence adapter; b) performing a single PCR cycle on the pWGAlib using a first primer (1PR) comprising from 5′ to 3′ a first sequencing adapter (1PR5SA) and a first primer 3′ section (1PR3S) hybridizing to the reverse complementary of the WGA library universal sequence adapter; c) performing a single PCR cycle on the on the product of step b) using a second primer (2PR) comprising from 5′ to 3′ a second sequencing adapter (2PR5SA) different from the 1PR5SA, and a second primer 3′ section (2PR3S) hybridizing to the WGA library universal sequence adapter reverse complementary; d) amplifying by PCR the product of step c) using a third primer comprising the 1PR5SA and a fourth primer comprising 2PR5SA.

There is disclosed a method of generating a massively parallel sequencing library comprising the steps of: a) providing a primary WGA DNA library (pWGAlib), including fragments comprising a WGA library universal sequence adaptor; b) re-amplifying the primary WGA DNA library using at least one first primer (1PR) and at least one second primer (2PR); the at least one first primer (1PR) comprising from 5′ to 3′ at least one first sequencing adaptor (1PR5SA), at least one first sequencing barcode (1PR5BC) and a first primer 3′ section (1PR3S) hybridizing to either the WGA library universal sequence adaptor or its reverse complementary; the at least one second primer (2PR) comprising from 5′ to 3′ at least one second sequencing adaptor (2PR5SA) different from the at least one first sequencing adaptor (1PR5SA), and a second primer 3′ section (2PR3S) hybridizing to either the WGA library universal sequence adaptor or its reverse complementary.

The invention relates to a kit and a method for concentrating fetal free DNA, and an application of the kit. DNA extracted from pregnant women peripheral blood or a mixture of placental tissues and the pregnant women peripheral blood is used as a DNA template, MSRE enzyme digestion is carried out, and an amplification primer is adopted to carry out RT-qPCR amplification in order to obtain a PCR product. Compared with kits in the prior art, the kit in the invention has the advantages of detection of the cffDNA of fetal specific trisomy 21st chromosome, being as low as 0.1%, and high sensitivity. The kit is simpler and cheaper than massive parallel sequencing detection of the trisomy 21 syndrome. The kit uses MSRE to process a gene, so the gene loss is less than that of sulfite gene treatment, and the diagnosis accuracy is improved.

This invention relates to methods, compositions and kits for extending a polynucleotide and for preparing sequencing library of polynucleotides involving generating modified target polynucleotide on an adaptor template oligonucleotide and tagging one or two strands of a target sequence. The sequencing library is suitable for massive parallel sequencing and comprises a plurality of double-stranded nucleic acid molecules.