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Samples typically consist of a mixture of genomes at widely varying abundance levels.
This may cause assignment of closely related sequences to the same reference genome, perhaps at a higher taxonomic level (ie, order or class), thereby generating bins containing a mixture of genomes.
Future investigation of all of these problems will be necessary if the goal of sequencing studies is to characterize all mutations present in the heterogeneous mixture of genomes that make up a tumor.
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The following potential advantage of NNMA over NGS further supports this approach: NNMA can deal with an organism as a whole, whereas NGS yields a mixture of genome fragments for analysis.
NGS methodologies comprehensively and systematically determine both nucleotide sequence and copy number of genomic loci, and have the advantage of being able to simultaneously sequence heterogeneous mixtures of genomes in a given sample, which might include tumour, stromal, and immune cells.
71, 72 Annotation of metagenomes is specifically designed to work with mixtures of genomes and contigs of varying length.
Assembly tools such as IDBA-UD are being developed and increasingly improved to address the specific problem of assembling mixtures of genomes as is eminent for metagenomic samples.
To discriminate mixtures of genomes at various dosages, the profiles of fragments and band intensities were initially established by sequential mixtures of DNA samples from the M. acuminata (AA; 'Calcutta 4') and the M. balbisiana (BB; 'Butuhan') genomes to obtain various artificial combinations of genomes.
These samples would be pooled and processed by using available technology to isolate virus particles en masse, recover viral nucleic acids, produce amplified shotgun libraries, carry out shotgun sequencing of the mixture of viral genomes, and reconstruct these genomes in silico with the techniques originally developed to sequence the entire human genome from random fragments.
These results show that, given an appropriate reference sequence for each genome under consideration, it is possible to assemble short reads from a mixture of mitochondrial genomes and deconvolute the resulting contigs without the need to index the reads.
The described techniques also evade protective measures against "heteroplasmy," the mixture of mitochondrial genomes that occasionally results from the persistence of the sperm mitochondrion after fertilization.
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