YY/T 1723-2020High-throughput gene sequencer (English PDF)

3.1 Sequencing

The determination of the different base types of nucleic acid molecules, that is, the determination of adenine (A), guanine (G), cytosine (C) and thymus

The composition or sequence of bases such as adenine (T) or uracil (U).

Note: Rewrite GB/T 30989-2014, definition 3.18.

3.2 Data output per run

The number of gene fragments for which sequence information can be obtained in a single sequencing or the deoxyribonucleic acid and ribonucleic acid (expressed in bases) that can be measured Quantity. [GB/T 30989-2014, definition 3.21]

3.3 Output per run

The number of gene fragments or bases that are qualified in a single run

Note: Rewrite GB/T 30989-2014, definition 3.23.

3.4 Read length

The length of a sequence fragment of acceptable quality that can be read in a single run, usually expressed in bases.

Note: Rewrite GB/T 30989-2014 and define 3.24.

3.5 Base calling

The process of converting fluorescent signals or other signals generated by the sequencing reaction into sequence information in the sequencing process.

[GB/T 30989-2014, definition 3.26]

3.6 Base quality value

A measure of the probability of the base being correctly identified. Usually expressed directly in ASCII code.

Note: Rewrite GB/T 30989-2014 and define 3.29.

3.7 High-throughput sequencing

The gene sequencer, which is obviously different from Sanger sequencing, is mainly characterized in that it is not necessary to pre-clear the sequence of the primers of the target fragments.

Columns, based on fragmented DNA, relying on independent reaction system for clonal amplification, capable of performing hundreds of thousands to millions of nucleic acid sequences at a time (DNA) The technical characteristics of parallel molecular sequence determination and generally shorter read lengths are signs.

Note: At present, high-throughput gene sequencers are roughly distinguished according to their throughput and suitability for clinical applications such as human genome, whole exome, and target genes.

Such as sequencing throughput >=20GB/run high-throughput gene sequencer, sequencing throughput ＜20GB/run and >=2GB/run high-throughput gene sequencer and measurement Sequence throughput< 2Gb/run and >=100Mb/run high-throughput gene sequencer.

4.1 Appearance requirements

The appearance of the instrument meets the following requirements.

--- The surface of the equipment should not have obvious bumps, scratches, paint stripping, flow marks, and metal parts should not be rusted and mechanically damaged;

---The switches and keys should be flexible and reliable, and the fasteners should be tight and not loose;

---Signs and symbols should be clear and correct.

4.2 Sequencing read length and throughput

Under the prescribed read length mode of sequencing, the sequencing throughput and the effective sequencing throughput should meet the requirements specified by the manufacturer.

Note: The sequencing read length mode is specified according to the performance requirements of the reagents used, such as double-ended 150 bp, single-ended.200 bp and so on.

4.3 The percentage of base recognition quality

Single sequencing, count the percentage of base recognition quality above the specified threshold, and the average percentage of base recognition quality should not be lower than the manufacturer Prescribed requirements.

Note: Sequencers with different principles have different expressions of base recognition quality, such as Q20, Q30, etc.

4.4 Performance Index

4.4.1.1 Sequencing coverage and average sequencing depth

The manufacturer shall specify the sequencing coverage and average sequencing depth of the national reference or standard products.

Note: Refer to Appendix A for the description of national reference products involved in this standard.

4.4.1.2 Sequencing accuracy rate

Under the sequencing coverage and average sequencing depth specified by the manufacturer, the following requirements are met.

a) For detection of human genome (DNA) reference or standard products, the comparison rate should meet the manufacturer's requirements, and should be consistent with single base variation (SNP), insertion Incoming deletion variant (Indel) reference data set comparison, the accuracy and sensitivity of SNP and Indel should meet the manufacturer's requirements;

b) For the detection of human genome DNA reference or standard products, the comparison rate should meet the manufacturer's requirements, and the human genome DNA reference sequence The 1GB reference sequence alignment is specified in the column, and the accuracy of sequencing consensus sequence should not be less than 99.0%;

c) Detect bacterial and viral DNA reference products and compare them with corresponding reference sequences. The accuracy of sequencing consensus sequences should not be less than 99.0%.

Note 1. For gene sequencers that are limited to the detection of human genome DNA, only the reference or standard product of human genome DNA can be tested, that is, a) and b) Detection.

Note 2. For gene sequencers that are limited to the detection of pathogenic microorganisms, only bacterial and viral DNA reference detections can be performed, that is, the detection of c).

Note 3. Gene sequencers for single-stranded sequence analysis can increase the detection indicators of haplotype genome DNA reference products.

4.4.1.3 Repeatability

Performing 3 repetitions of sequencing can meet the requirements of 4.4.1.1 and 4.4.1.2.

4.4.2.1 Sequencing coverage and average sequencing depth

The manufacturer shall specify the sequencing coverage and average sequencing depth of the national reference or standard products.

4.4.2.2 Sequencing accuracy rate

Under the sequencing coverage and average sequencing depth specified by the manufacturer, the following requirements are met.

a) Detect the all-exon region specified in the human genome DNA reference product or standard product, and the comparison rate should meet the manufacturer's requirements, and For comparison of the designated SNP and Indel reference data sets for all exon regions, the accuracy and sensitivity of SNP and Indel should be in line with the manufacturer The requirements;

b) Detect all exon regions specified in the DNA reference product or standard product of the human genome. The comparison rate should meet the manufacturer's requirements.

Alignment of the entire exon region specified in the DNA reference sequence of the human genome, and the accuracy of the sequencing consensus sequence should not be less than 99.0%;

c) Detect bacterial and viral DNA reference products and compare them with corresponding reference sequences. The accuracy of sequencing consensus sequences should not be less than 99.0%.

Note 1. For gene sequencers that are limited to the detection of human genome DNA, only the reference or standard product of human genome DNA can be tested, that is, a) and b) Detection.

Note 2. For gene sequencers that are limited to the detection of pathogenic microorganisms, only bacterial and viral DNA reference detections can be performed, that is, the detection of c).

Note 3. Gene sequencers for single-stranded sequence analysis can increase the detection indicators of haplotype genome DNA reference products.

4.4.2.3 Repeatability

Performing three repetitions of sequencing should all meet the requirements of 4.4.2.1 and 4.4.2.2.

4.4.3.1 Sequencing coverage and average sequencing depth

The manufacturer shall specify the sequencing coverage and average sequencing depth of the national reference or standard products.

4.4.3.2 Sequencing accuracy rate

Under the sequencing coverage and average sequencing depth specified by the manufacturer, the following requirements are met.

a) To detect the 17Kb region specified in the human genome DNA reference product or standard product, the comparison rate should meet the manufacturer's requirements, and Determine the 10 designated SNP sites in the 17Kb area, and the accuracy of SNP detection should not be less than 90%;

b) Detect the sequence alignment of the designated 17Kb region in the human genome DNA reference product or standard product, and the accuracy of the sequencing consensus sequence should Not less than 99.0%;

c) Detect bacterial and viral DNA reference products and compare them with corresponding reference sequences. The accuracy of sequencing consensus sequences should not be less than 99.0%.

Note 1. For gene sequencers that are limited to the detection of human genome DNA, only the reference or standard product of human genome DNA can be tested, that is, a) and b) Detection.

Note 2. For gene sequencers that are limited to the detection of pathogenic microorganisms, only bacterial and viral DNA reference detections can be performed, that is, the detection of c).

Note 3. Gene sequencers for single-stranded sequence analysis can increase the detection indicators of haplotype genome DNA reference products.

4.4.3.3 Repeatability

Performing three repetitions of sequencing should all meet the requirements of 4.4.3.1 and 4.4.3.2.

4.5 Software functions

The software function of a high-throughput gene sequencer should include at least two parts. the instrument operation control function and the signal real-time acquisition function.

a) The instrument operation control software should be able to guide the completion of instrument operation and control, and meet the manufacturer's requirements; […]