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6 QC methods post library construction for NGS

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After nucleic acid extraction and sample QC, the next step in the NGS workflow is library preparation. NGS libraries are prepared to meet the platform requirements with respect to size, purity, concentration and efficient ligation of adaptors. Assessing the quality of a sequencing library before committing it to a full-scale sequencing run ensures maximum sequencing efficiency, leading to accurate sequencing data with more even coverage.

In this blog post, we list the various ways to QC libraries in order of most stringent to least stringent.

1. qPCR

qPCR is a method of quantifying DNA based on PCR. qPCR tracks target concentration as a function of PCR cycle number to derive a quantitative estimate of the initial template concentration in a sample. As with conventional PCR, it uses a polymerase, dNTPs, and two primers designed to match sequences within a template. For the QC protocol, the primers match sequences within the adapters flanking a sequencing library.

Therefore, qPCR is an ideal method for measuring libraries in advance of generating clusters, because it will only measure templates that have both adaptor sequences on either end which will subsequently form clusters on a flow cell. In addition, qPCR is a very sensitive method of measuring DNA and therefore dilute libraries with concentrations below the threshold of detection of conventional spectrophotometric methods can be quantified by qPCR.

KAPA Biosystems SYBR FAST ‘Library Quantification Kit for Illumina Sequencing Platforms is commonly used with qPCR. This kit measures absolute numbers of molecules containing the Illumina adapter sequences, thus providing a highly accurate measurement of amplifiable molecules available for cluster generation.

2. MiSeq

The MiSeq system uses the same library prep methods and proven sequencing by synthesis chemistry as the HiSeq system. Thus, it is ideal for analyzing prepared libraries prior to performing high-throughput sequencing. Performing library quality control (QC) using the MiSeq system before committing it to a fullscale HiSeq run can save time and money while leading to better sequencing results.

Data generated by the MiSeq system is comparable to other Illumina next-generation sequencing platforms, ensuring a smooth transition from one instrument to another. Based on the individual experimental requirements, metrics obtained from performing simple QC can be used to streamline and improve your sequencing projects.

Using a single library prep method and taking only a single day, detailed QC parameters, including cluster density, library complexity, percent duplication, GC bias, and index representation can be generated on the MiSeq system. The MiSeq system has the unique ability to do paired-end (PE) sequencing for accurately assessing insert size. Library cluster density can also be determined and used to predict HiSeq cluster density, maximizing yield and reducing rework.

3. Fluorometric method

Quantifying DNA libraries using a fluorometric method that involves intercalating dyes specifically binding to DNA or RNA is highly useful. This method is very precise as DNA dyes do not bind to RNA and vice versa.

The Invitrogen™ Qubit™ Fluorometer a popular fluorometer that accurately measures DNA, RNA, and protein using the highly sensitive Invitrogen™ Qubit™ quantitation assays. The concentration of the target molecule in the sample is reported by a fluorescent dye that emits a signal only when bound to the target, which minimizes the effects of contaminants—including degraded DNA or RNA—on the result.

4. Automated electrophoresis

Several automated electrophoretic instruments are useful in estimating the size of the NGS libraries. The Agilent 2100 Bioanalyzer system provides sizing, quantitation, and purity assessments for DNA, RNA, and protein samples. The Agilent 2200 TapeStation system is a tape-based platform for reliable electrophoresis platform for accurate size selection of generated libraries. PerkinElmer LabChip GX can be used for DNA and RNA quantitation and sizing using automated capillary electrophoresis separation. The Qiagen QIAxcel Advanced system fully automates sensitive, high-resolution capillary electrophoresis of up to 96 samples per run that can be used for library QC as well. All these instruments are accompanied by convenient analysis and data documentation software that make the library QC step faster and easier.

5. UV-Visible Spectroscopy

A UV-Vis spectrophotometer can be used to analyze spectral absorbance to measure the nucleic acid libraries and can differentiate between DNA, RNA and other absorbing contaminants. However, this method is not super accurate and should be paired with one of the other QC methods to ensure high-quality libraries. There are several US-Vis spectrophotometers currently available, such as currently available such as Thermo Scientific™ NanoDrop™ UV-Vis spectrophotometer, Qiagen QIAExpert System, Shimadzu Biospec-nano etc.

6. Bead normalization

This is the preferred QC method if < 12 libraries are to be QCed or if library yields are less than 15 nM, highly variable and unpredictable or Users are working with uncharacterized genomes and are inexperienced with the Nextera XT DNA Library Prep Kit protocol.

During bead-based normalization, DNA is bound to normalization beads and eluted off the beads at approximately the same concentration for each sample. Bead-based normalization enables scientists to bypass time-consuming library quantitation measurements and manual pipetting steps before loading libraries onto the sequencer. Bead-based normalization can provide significant cost and time savings for researchers processing many samples, or for researchers without access to any of the QC  instruments listed in the above methods.

 

 

 

 

Top 3 Sample QC steps prior to library preparation for NGS

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Before beginning library preparation for next-generation sequencing, it is highly recommended to perform sample quality control (QC) to check the nucleic acid quantity, purity and integrity. The 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, RNA, PCR amplicons, and ChIP samples, must have high purity and integrity and sufficient concentration for the sequencing reaction.

1. Nucleic Acid Quantification

Measuring the concentration of nucleic acid samples is a key QC step to determine the fit and amount of nucleic acid available for further processing.

  • Absorbance Method:

A UV-Vis spectrophotometer can be used to analyze spectral absorbance to measure the whole nucleic acid profile and can differentiate between DNA, RNA and other absorbing contaminants. Different molecules such as nucleic acids, proteins, and chemical contaminants absorb light in their own pattern. By measuring the amount of light absorbed at a defined wavelength, the concentration of the molecules of interest can be calculated. Most laboratories are equipped with a US-Vis spectrophotometer to quantify nucleic acids or proteins for their day-to-day experiments. Customers can choose from several spectrophotometers currently available such as Thermo Scientific™ NanoDrop™ UV-Vis spectrophotometer, Qiagen QIAExpert System, Shimadzu Biospec-nano etc.

  • Fluorescence Method:

Fluorescence methods are more sensitive than absorbance, particularly for low-concentration samples, and the use of DNA-binding dyes allows more specific measurement of DNA than spectrophotometric methods. Fluorescence measurements are set at excitation and emission values that vary depending on the dye chosen (Hoechst bis-benzimidazole dyes, PicoGreen® or QuantiFluor™ dsDNA dyes). The concentration of unknown samples is calculated based on comparison to a standard curve generated from samples of known DNA concentration.

The availability of single-tube and microplate fluorometers gives flexibility for reading samples in PCR tubes, cuvettes or multiwell plates and makes fluorescence measurement a convenient modern alternative to the more traditional absorbance methods. Thermo Scientific (Invitrogen) Qubit™ Fluorometer is one of the most commonly used fluorometers that accurately measure low concentration DNA, RNA, and protein.

sho-qubit-instrument

2. Nucleic Acid Purity

Nucleic acid samples can become contaminated by other molecules with which they were co-extracted and eluted during the purification process or by chemicals from upstream applications. Purification methods involving phenol extraction, ethanol precipitation or salting-out may not completely remove all contaminants or chemicals from the final eluates. The resulting impurities can significantly decrease the sensitivity and efficiency of your downstream enzymatic reactions.

  • UV spectrophotometry measurements enable calculation of nucleic acid concentrations based on the sample’s absorbance at 260 nm. The absorbance at 280 nm and 230 nm can be used to assess the level of contaminating proteins or chemicals, respectively. The absorbance ratio of nucleic acids to contaminants provides an estimation of the sample purity, and this number can be used as acceptance criteria for inclusion or exclusion of samples in downstream applications.
  • 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/A280ratio, 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 Qiagen QIAxpert system enables spectral content profiling, which can discriminate DNA and RNA from sample contaminants without using a dye.

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  • qPCR:

Quantitative PCR, or real-time PCR, (qPCR) uses the linearity of DNA amplification to determine absolute or relative quantities of a known sequence in a sample. By using a fluorescent reporter in the reaction, it is possible to measure DNA generation in the qPCR assay. In qPCR, DNA amplification is monitored at each cycle of PCR. When the DNA is in the log-linear phase of amplification, the amount of fluorescence increases above the background. The point at which the fluorescence becomes measurable is called the threshold cycle (CT) or crossing point. By using multiple dilutions of a known amount of standard DNA, a standard curve can be generated of log concentration against CT. The amount of DNA or cDNA in an unknown sample can then be calculated from its CT value.

qPCR-based assays can accurately qualify and quantify amplifiable DNA in challenging samples. For example, DNA derived from Formalin-fixed paraffin-embedded tissue samples, is oftentimes highly fragmented, cross-linked with protein and has a high proportion of single-stranded DNA making it challenging to perform library preparation steps. For FFPE samples, the Agilent NGS FFPE QC kit enables functional DNA quality assessment of input DNA.

3. Nucleic Acid Integrity (Size distribution)

Along with quantity and purity, size distribution is a critical QC parameter that provides valuable insight into sample quality. Analyzing nucleic acid size informs you about your sample’s integrity and indicates whether the samples are fragmented or contaminated by other DNA or RNA products. Various electrophoretic methods can be used to assess the size distribution of your sample.

  • Agarose Gel Electrophoresis

In this method, a horizontal gel electrophoresis tank with an external power supply, analytical-grade agarose, an appropriate running buffer (e.g., 1X TAE) and an intercalating DNA dye along with appropriately sized DNA standards are required. A sample of the isolated DNA is loaded into a well of the agarose gel and then exposed to an electric field. The negatively charged DNA backbone migrates toward the anode. Since small DNA fragments migrate faster, the DNA is separated by size. The percentage of agarose in the gel will determine what size range of DNA will be resolved with the greatest clarity. Any RNA, nucleotides, and protein in the sample migrate at different rates compared to the DNA so the band(s) containing the DNA will be distinct.

gel_electrophoresis_dna_bands_yourgenome

Analyzing PCR amplicons or RFLP fragments confirms the presence of the expected size fragments and alerts you to the presence of any non-specific amplicons. Electrophoresis also helps you assess the ligation efficiency yield for plasmid cloning procedures as well as the efficiency of removal of primer–dimers or other unspecific fragments during sample cleanup.

For complex samples such as genomic DNA (gDNA) or total RNA, the shape and position of the smear from electrophoresis analysis directly correlates with the integrity of the samples. Nucleic acid species of larger size tend to be degraded first and provide degradation products of lower molecular weight. Samples of poor integrity generally have a higher abundance of shorter fragments, while high-quality samples contain intact nucleic acid molecules with higher molecular size.

Eukaryotic RNA samples have unique electrophoretic signatures, which consist of a smear with major fragments corresponding to 28S, 18S and 5S ribosomal RNA (rRNA). These electrophoretic patterns correlate with the integrity of the RNA samples. The RNA integrity can either be assessed manually or with automation that employs a dedicated algorithm such as the RNA Integrity Number (RIN) that gives an objective integrity grade to RNA samples ranging from 1–10. RNA samples of highest quality usually have a score of 8 or above.

  • Capillary Electrophoresis

In this method, charged DNA or RNA molecules are injected into a capillary and are resolved during migration through a gel-like matrix. Nucleic acids are detected as they pass by a detector that captures signals of specific absorbance. Results are presented in the form of an electropherogram, which is a plot of signal intensity against migration time. The fragment sizes are precisely determined using a size marker consisting of fragments of known size. This method provides highly resolving and sensitive nucleic acid analysis that is faster and safer.