The quality of the NGS library is the factor with the most influence on the success of the sequencing run, affecting both the sequence validity and the number of reads. QC procedures tracking success of library preparation steps ensure that only samples of good quality are processed downstream and sequenced to generate reads of highest quality that can be confidently turned into insights. After sequencing and variant analysis, results need to be verified and validated using a technology other than NGS, such as Pyrosequencing.
Step 1: Sample preparation
Regardless of the origin of the starting material, successful sequencing experiments require purification of high-quality nucleic acids.
Although automated nucleic acid extraction methods provide high throughput, reproducibility and yield, it is still recommended to perform QC checks of quantity, purity and integrity of the samples prior to processing. Starting material for NGS library construction might be any type of nucleic acid that is or can be converted into double-stranded DNA (dsDNA). These materials, often gDNA, PCR amplicons, ChIP samples and RNA, must have high purity and integrity and sufficient concentration for the sequencing reaction.
Contaminants such as RNA, proteins or chemicals can interfere with library preparation and the sequencing reactions. When sequencing DNA, an RNA removal step is highly recommended, and when sequencing RNA, a gDNA removal step is recommended. Sample purity can be assessed following nucleic acid extraction and throughout the library preparation workflow using UV/Vis spectrophotometry. For DNA and RNA samples the relative abundance of proteins in the sample can be assessed by determining the A260/A280 ratio, which should be between 1.8–2.0. Contamination by organic compounds can be assessed using the A260/A230 ratio, which should be higher than 2.0 for DNA and higher than 1.5 for RNA. Next-generation spectrophotometry with the QIAxpert system enables spectral content profiling, which can discriminate DNA and RNA from sample contaminants without using a dye.
Although automated nucleic acid extraction methods provide high throughput, reproducibility and yield, it is still recommended to perform QC checks of quantity, purity and integrity of the samples prior to processing. Starting material for NGS library construction might be any type of nucleic acid that is or can be converted into double-stranded DNA (dsDNA). These materials, often gDNA, PCR amplicons, ChIP samples and RNA, must have high purity and integrity and sufficient concentration for the sequencing reaction.
Contaminants such as RNA, proteins or chemicals can interfere with library preparation and the sequencing reactions. When sequencing DNA, an RNA removal step is highly recommended, and when sequencing RNA, a gDNA removal step is recommended. Sample purity can be assessed following nucleic acid extraction and throughout the library preparation workflow using UV/Vis spectrophotometry. For DNA and RNA samples the relative abundance of proteins in the sample can be assessed by determining the A260/A280 ratio, which should be between 1.8–2.0. Contamination by organic compounds can be assessed using the A260/A230 ratio, which should be higher than 2.0 for DNA and higher than 1.5 for RNA. Next-generation spectrophotometry with the QIAxpert system enables spectral content profiling, which can discriminate DNA and RNA from sample contaminants without using a dye.
Step 2: Target enrichment and library preparation
After purification, nucleic acids must be processed through the NGS library preparation workflow in order to be able to meet the platform requirements with respect to size, purity, concentration and efficient ligation of adaptors (refer to the QIAseq portfolio for more details on library preparation).
First, nucleic acids are sheared into smaller fragments using either physical or enzymatic shearing methods. A particular size distribution of the sheared samples is required for successful sequencing, because NGS platforms are optimized to sequence fragments within a given size range. Incomplete shearing or excessive shearing generates fragments that are too long or too short and are sub-optimal for the sequencing reaction.
Since sequencers require DNA templates, RNA samples must be reverse transcribed into cDNA prior to further processing. DNA is more stable than RNA, so this step also increases the stability of the samples without compromising the genetic information they carry. Fragmented DNA or cDNA fragments must undergo end repair and adenylation to ligate the specific adaptors required by the sequencing platform. Following ligation, size distribution and concentration analysis are performed to check the success of library preparation steps and assess the quality of the library before the sequencing reaction.
The library may also undergo specific PCR amplification of regions of particular interest to increase the sequencing depth of these regions. Libraries prepared without enrichment procedures have reduced library preparation bias but are more challenging to assess for QC. The sequencing depth of areas such as GC-rich regions, promoters and repeat regions is improved, thus leveraging the detection and discovery of new sequence variants.
Following library amplification, size selection steps remove all contaminants, such as unligated adapters and primer–dimers as well as fragments that are too short or too long. Size distribution and concentration measurement using a capillary electrophoresis system such as the QIAxcel Advanced, for example, is the final QC step prior to the sequencing run to ensure the library has been correctly amplified and purified.
First, nucleic acids are sheared into smaller fragments using either physical or enzymatic shearing methods. A particular size distribution of the sheared samples is required for successful sequencing, because NGS platforms are optimized to sequence fragments within a given size range. Incomplete shearing or excessive shearing generates fragments that are too long or too short and are sub-optimal for the sequencing reaction.
Since sequencers require DNA templates, RNA samples must be reverse transcribed into cDNA prior to further processing. DNA is more stable than RNA, so this step also increases the stability of the samples without compromising the genetic information they carry. Fragmented DNA or cDNA fragments must undergo end repair and adenylation to ligate the specific adaptors required by the sequencing platform. Following ligation, size distribution and concentration analysis are performed to check the success of library preparation steps and assess the quality of the library before the sequencing reaction.
The library may also undergo specific PCR amplification of regions of particular interest to increase the sequencing depth of these regions. Libraries prepared without enrichment procedures have reduced library preparation bias but are more challenging to assess for QC. The sequencing depth of areas such as GC-rich regions, promoters and repeat regions is improved, thus leveraging the detection and discovery of new sequence variants.
Following library amplification, size selection steps remove all contaminants, such as unligated adapters and primer–dimers as well as fragments that are too short or too long. Size distribution and concentration measurement using a capillary electrophoresis system such as the QIAxcel Advanced, for example, is the final QC step prior to the sequencing run to ensure the library has been correctly amplified and purified.
Step 3: Validation
After the sequencing run, variant calling, and narrowing down regions of interest that link a genotype to an observed phenotype, the results must be verified and validated using an alternate sequencing technology, such as Pyrosequencing. It is highly recommended to use an alternative technology, due to the high risk of false-positive variants in the NGS workflow.
Find out more about QC checks in other laboratory workflows:
References
NGS comes with many challenges that scientists must address. Defining guidelines and sharing best practice help guide researchers new to NGS and ensure the quality and relevance of the NGS results when shared. This selection of articles and reviews provide resources for assay design, quality assurance, quality control, data management and results analysis and interpretation.
- Matthijs, G. et al. 2016. Guidelines for next-generation sequencing. European Journal of Human Genetics. 24, 2–5.
- Rehm, H.L. et al. 2013. ACMG clinical laboratory standards for next-generation sequencing. Genetics in medicine 15:9, 733–747.
- Mack, S.J. et al. 2015. Minimum information for reporting next generation sequence genotyping (MIRING): Guidelines for reporting HLA and KIR genotyping via next generation sequencing. Human Immunology. 76:12, 954–962.
- Ellard, S. et al. Practice guidelines for targeted next generation sequencing analysis and interpretation. Association for Clinical Genetic Science. Updated May 2014.