PCR Sequencing via DNA Incomplete Shearing for HLA Genotyping

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Solution Overview

Problem

Current second-generation DNA sequencing techniques, such as Illumina GA and Roche 454, are limited by short maximum sequencing lengths, leading to incomplete sequencing of PCR amplicons and high costs, which restrict their application in large-scale HLA genotyping and other fields.

Innovation Solution

The method employs a combination of primer indexing, DNA incomplete shearing, and PCR-Free library preparation to extend the sequenced length of PCR products beyond the maximum sequencing length of the sequencer, utilizing the high throughput and low cost of second-generation sequencing techniques, and applies this to HLA genotyping using Illumina GA sequencing.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If second-generation DNA sequencing technique is used, then sequencing throughput is improved and cost is reduced, but maximum sequencing length is limited to short range

Engineering Contradiction:

Improvesequencing throughputVSAvoidmaximum sequencing length

Core Design Contradiction:

ProductivityVSLength of stationary object

Solution Approach 1:

The patent divides the PCR amplicon into multiple overlapping fragments through incomplete shearing, where each fragment is sequenced individually. The sequencing reads are then assembled computationally to reconstruct the complete sequence, thereby overcoming the short read length limitation while maintaining high throughput and low cost of second-generation sequencing

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent transitions from direct sequencing of long amplicons to a two-dimensional approach: first fragmenting the DNA into shorter segments that can be sequenced, then assembling these segments through computational overlap analysis. This dimensional transformation enables sequencing of long targets using short-read technology

Inventive Principle:

Principle #17Another dimension (Dimensionality change)

2Measurement precision

If PCR amplicon length is greater than maximum sequencing length, then complete sequencing cannot be accomplished, but sequencing coverage is reduced

Engineering Contradiction:

Improvesequencing completenessVSAvoidsequencing coverage

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The patent performs preliminary incomplete shearing of the PCR amplicon into overlapping fragments before sequencing. This pre-processing step ensures that all subsequent sequencing reads can be assembled to cover the entire amplicon, guaranteeing complete sequencing coverage even though individual reads are short

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent uses computational assembly methods that continuously overlap and stitch together multiple short sequencing reads to reconstruct the complete amplicon sequence. This continuous computational processing ensures that no gaps are introduced, maintaining 100% sequencing coverage throughout the entire target region

Inventive Principle:

Principle #20Continuity of useful action

Data Source

PatentUS9957564B2Application of a PCR sequencing method, based on DNA barcoding technique and DNA incomplete shearing strategy, in HLA genotyping

Publication Date: 2018.05.01 BGI GENOMICS CO LTD

AI summary

The invention provides a PCR sequencing method, wherein the combination of primer indexes, DNA incomplete shearing strategy and the second generation sequencing technique (Paired-End sequencing technique) can make the length of PCR products that can be sequenced by a sequencer longer than the maximum sequencing length of the sequencer while making full use of the characteristics of the second generation sequencing technique such as high throughput and low cost, thereby greatly broadening its applicable scope. In addition, the present invention also provides primer indexes for the PCR sequencing method and the use of the method in genotyping, particularly in HLA analysis, and also provides the PCR primers used, particularly the PCR primers for HLA-A, B, HLA-C and HLA-DQB1 gene.