Position anchoring bar code system for nanopore sequencing library building

A technology for nanopore sequencing and sequencing library, which is applied in the field of position-anchored barcode system, can solve the problems of doctor's diagnosis interference, poor metagenomic species analysis accuracy, barcode confusion and other problems, so as to screen out long-distance barcode interference and reduce false The effect of positive identification and result accuracy improvement

A technology for nanopore sequencing and sequencing library, which is applied in the field of position-anchored barcode system, can solve the problems of doctor's diagnosis interference, poor metagenomic species analysis accuracy, barcode confusion and other problems, so as to screen out long-distance barcode interference and reduce false The effect of positive identification and result accuracy improvement

CN111440846AActive Publication Date: 2020-07-24SIMCERE DIAGNOSTICS CO LTD +1
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  • Position anchoring bar code system for nanopore sequencing library building
  • Position anchoring bar code system for nanopore sequencing library building
  • Position anchoring bar code system for nanopore sequencing library building

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1 comparison error statistics

[0082] The present invention considers that the main cause of barcode confusion is the sequence difference between the sequenced barcode and the preset real barcode, so the difference between the sequenced barcode sequence and the real barcode sequence is firstly sorted out. For this reason, the present invention uses 10 sets of barcodes of the SQK-PBK004 kit of ONT Company to separately build a library for the sample DNA on the computer, intercept the 250 bp of the 5' end of the sequencing data to ensure that the barcode region is included, and then perform global analysis with the corresponding preset barcode adapter Comparison (overlap alignment). Finally, according to the output multiple alignment file, the alignment difference of each position of the barcode adapter sequence is summarized and sorted, and the error position distribution and error type of the sequencing barcode are counted. Among them, the error types are d...

Embodiment 2

[0090] Embodiment 2 The impact of different error types and barcode lengths on the overall accuracy of barcode comparison

[0091] According to the different error types and their corresponding error rate values ​​in the above-mentioned embodiment 1, the present invention respectively simulates barcode sequences with lengths of 20bp, 40bp, 60bp, 80bp, 100bp, and 120bp in total, and each group simulates 12 different barcode elements . The present invention takes 80bp as an example to illustrate the specific overview of the simulation. First, the present invention presets 12 ideal barcode sequences, and the sequence information is shown in Table 2:

[0092] Table 2.

[0093]

[0094]

[0095] Then three different types of errors were introduced with the probability of a total error rate of 0.08 for each site, which were only indels, only base mismatches, and both indels and base mismatches. The detailed error rate distribution ratio of each error type is consistent with...

Embodiment 3

[0097] Example 3 The impact of inserting anchor sequences on the overall accuracy of barcode alignment

[0098] Based on the conclusion of the above-mentioned embodiment 2, the present invention intends to modify the barcode fragment in the following two ways, taking 80bp as an example:

[0099] 1) 1 anchor sequence insertion: replace the 12 bases in the middle of the barcode sequence with an anchor sequence with the same 12bp sequence (as shown in the underline in Table 2), that is, replace the original preset barcode fragment with the same length "short barcode- Anchor sequence-short barcode” format;

[0100] 2) Insertion of 2 anchor sequences: replace the 20 bp at both ends of the barcode sequence in Table 2 with 12 bp anchor sequence 1 and anchor sequence 2 (as shown in bold in Table 2), that is, short barcodes of the same length- Anchor Sequence 1 - Short Barcode - Anchor Sequence 2 - Short Barcode.

[0101] The location information of the anchor sequences obtained duri...

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Abstract

The invention relates to a position anchoring bar code system for nanopore sequencing library building, and a preparation method and application of the position anchoring bar code system. The positionanchoring bar code system provided by the invention has higher resolution and higher classification accuracy, can significantly reduce the false positive rate of identification, improves the nanoporesequencing precision on the whole, and reduces the sequencing cost.

Description

technical field [0001] The invention relates to the field of gene sequencing, in particular to a position-anchored barcode system for nanopore sequencing library construction. Background technique [0002] At present, there are many clinically infected patients in the world and there are various sources of infection. In China, infectious diseases even account for 49% of the total incidence of all diseases. At present, the conventional clinical diagnosis method is the doctor's empirical judgment plus microscopic examination, biochemical analysis, etc. to determine the source of infection of the symptoms, but human factors, detection cycle and detection range limitations can easily lead to false detection and missed judgment, especially not conducive to acute Diagnosis and treatment of infection. With the vigorous development of high-throughput sequencing and genomics, metagenomic sequencing technology is flourishing in the field of infection diagnosis because it can quickly,...

Claims

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Application Information

Patent Timeline
24 Jul 2020
Publication
CN111440846A
IPC
C12Q1/6806; C12N15/10; C40B50/06; C40B70/00; C40B80/00
CPC
C12N15/1093; C12Q1/6806; C40B50/06; C40B70/00; C40B80/00; C12Q2565/631; C12Q2563/185; C12Q2525/191
Inventors
戴岩; 胡龙