Genomic selection and sequencing using encoded microcarriers

Abstract

The present invention relates to a method for determining the sequence of a nucleic molecule. Herein a capture oligonucleotide probe is attached to an encoded microcarrier, wherein the code of said microcarrier identifies the sequence of said oligonucleotide probe. The capture oligonucleotide probe is hybridized with a sample comprising nucleic acids molecules, wherein said DNA fragment comprises a sequence which is complementary to the sequence of the capture oligonucleotide probe. The sequence of the DNA molecule is determined, wherein the capture oligonucleotide probe serves as a primer for a DNA polymerase, in the case of single molecule sequencing this is a sequencing primer. After the sequence determination, the nucleotide sequence of the capture oligonucleotide probe is identified by determining the code on the microcarrier, which corresponds with the capture oligonucleotide probe. This sequence information directly identifies the location of the sequenced DNA fragment on the genome, allowing direct comparison.

Description

field of invention [0001] The present invention relates to nucleic acid hybridization and sequencing methods. The invention further relates to the selection, sequencing and identification of nucleic acid fragments using encoded microcarriers. Background of the invention [0002] Recent sequencing technologies allow the simultaneous determination of large numbers of nucleotide sequences. This eliminates the need to perform separate sequencing reactions in different capillaries or separate reaction wells. Typically, DNA samples are fragmented by mechanical or enzymatic techniques, after which individual DNA fragments are bound to a matrix via a type of nucleotide linker molecule attached to the fragments ( E.g walls of reaction chambers or microcarriers / beads), the nucleotide linker molecule also functions as a universal primer. For techniques that sequence more than a single molecule, the PCR-based amplification steps are as follows. For example, sequencing technologies s...

AI Technical Summary

Problems solved by technology

[0004] A disadvantage of current sequencing methods is the relatively short individual read length of the DNA that can be sequenced, resulting in sequences with limited information content

This makes it often difficult to locate the determined sequence in the reference genome sequence (sequence annotation)

Particularly difficult is the case of bisulfate sequencing, where unmethylated C nucleotides in the CpG sequence of a fragment are converted to T nucleotides prior to the sequencing reaction, resulting in The reduced information of the code alignment and thus the increased difficulty in the correct assembly of the final nucleic acid sequence

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