Table 1.

Comparison of studies related to NGS quantification

Previous studies related to NGS quantification	Internal standard sequence design	Quantitative method	Applications	Possibilities for applications in clinical mNGS	Refs
1	Use of genome sequences of natural species (marine microorganisms)	Relative quantification	Faecal microorganisms/mNGS	The inserted sequence may have the same fragment in the clinical sample	[16]
2	Use of 16S rRNA or ARGs sequences from natural species*	Absolute quantification	Environmental microorganisms/mNGS	The inserted sequence may have the same fragment in the clinical sample	[19]
3	Inverting the genome sequences of natural species	Relative quantification	Environmental microorganisms/mNGS	The inserted sequence may have the same fragment in the clinical sample	[18]
4	Commercial plasmid sequences were used	Relative quantification	Marine microorganism/metatranscriptome	Plasmids may cause contamination in clinical applications	[17]
5	Synthesis based on 16S rRNA with ITS and other primer segment sequences	Absolute quantification	Environmental microorganisms/tNGS†	Methodology based on amplicon sequencing, not applicable to mNGS	[20]
6	No insertion of internal standards (directly combined with ddPCR)	Absolute quantification	Oral and digestive tract microorganisms /tNGS	Methodology based on amplicon sequencing, not applicable to mNGS	[21]
This study	Multiple double-stranded DNA sequences not found in nature were designed and artificially synthesised to be inserted at different concentration gradients	Absolute quantification	Clinical pathogenic microorganisms/mNGS	Sequences designed and synthesised to ensure that there are no duplicates in clinical samples, in addition to the ability to quantify all targets in the sample.	/

*ARGs: antibiotic resistance genes.

†tNGS：targeted Next-Generation Sequencing.