Synthetic PhiX Sequences for NGS Error Correction

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Solution Overview

Problem

Next-generation sequencing platforms, such as Illumina's, face challenges with sequencing accuracy due to biases like phasing, signal decay, and cross-talk, which can lead to errors in DNA library preparation and sequencing, particularly in clinical applications where mutation rates may be low and close to error rates, necessitating a method to profile and reduce sequencing error rates.

Innovation Solution

The method involves using synthetic PhiX types (PhiX L and PhiX S) to profile sequencing error rates from library preparation to sequencing outcome, by designing specific sequences, amplifying and sequencing these synthetic PhiX types alongside target DNA molecules on patterned flow cells, and analyzing error profiles to enhance mutation call accuracy.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional sequencing methods are used, then sequencing throughput is improved, but sequencing accuracy deteriorates due to biases like phasing, signal decay, and cross-talk

Engineering Contradiction:

Improvesequencing throughputVSAvoidsequencing accuracy

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent introduces PhiX control sequences as an intermediary reference material that is sequenced alongside target DNA. These control sequences serve as a mediator to establish baseline error profiles and distinguish systematic sequencing errors from true biological variants, thereby improving accuracy without reducing throughput

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent implements a feedback mechanism where error profiles are generated from PhiX control sequences and used to correct or filter variants called from target DNA sequences. This feedback loop allows the system to learn and compensate for systematic errors, improving measurement precision while maintaining high throughput

Inventive Principle:

Principle #23Feedback

2Measurement precision

If sequencing depth is increased to improve accuracy, then error detection capability is improved, but the ability to detect low-frequency mutations deteriorates as error rates approach mutation rates

Engineering Contradiction:

Improveerror detection capabilityVSAvoidlow-frequency mutation detection

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

PhiX control sequences act as an intermediary reference that provides a separate channel for characterizing error modes. By analyzing errors in the control sequences, the system builds error profiles that can be applied to distinguish true low-frequency mutations from sequencing artifacts in target sequences

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent performs preliminary error profiling using PhiX control sequences before analyzing target DNA variants. This preliminary action establishes baseline error rates and patterns, enabling more reliable detection of low-frequency mutations by comparing against the pre-established error profile

Inventive Principle:

Principle #10Preliminary action

Data Source

PatentUS20230105122A1Error corrected method mitigates systematic error via sequencing DNA data of the surrounding flow cells of the variants on Patterned Flow Cell

Publication Date: 2023.04.06 KUO SHOU-CHIEN

AI summary

A method of determining a target nucleic acid of interest using synthetic Phix sequences designed to match the target nucleic acid fragments. The sequencing error profile was generated using the synthetic Phix L and synthetic Phix S, and the sequencing read locations information on the patterned flow cell.