6 QC methods post library construction for NGS

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 Next Generation Sequencing Applications

A common question we’re asked is what library preparation applications are researchers most interested in. Providers starting their own core facility, bioinformaticians writing software for a particular pipeline and others trying to gauge demand for NGS applications are most interested in this answer. In the last three months we looked at the number of initiated projects on Genohub that included library preparation. Projects initiated on Genohub are made through our Shop by Project: https://genohub.com/shop-by-next-gen-sequencing-project/ or our Shop by Technology: https://genohub.com/shop-by-next-gen-sequencing-technology/ interfaces. Users enter project information like coverage or the number of required reads and can specify if they prefer one platform over another. Genohub’s intelligent project matching engine takes this data and displays packages that consist of provider services that match the user’s request. Users who select a package and begin direct communication with the provider are considered those who have initiated a project. A summary of the library preparation applications those users choose in the projects started between 10/2013 and 12/2013 are plotted in Figure 1 (data of projects using our complementary consultation service was also included in this graph).  

projects started

RNA-Seq projects encompass all those starting with Total RNA, ribosomal depleted and poly-A select RNA. These applications were the most popular followed by projects involving whole genome sequencing. RNA-Seq’s growing versatility as both an expression analysis and de novo assembly/construction tool are likely the reasons for the greatest number of projects on Genohub. Targeted DNA applications were also frequently performed as Exome, 16S V4 and other Amplicon-Seq projects consisted of the 3rd, 4th and 5th most commonly started projects on Genohub. While not illustrated in Figure 1, specialized applications related to Methyl-Seq and ChIP-Seq were some of the fastest growing.

Having recently started, we expect these numbers to grow significantly. We’ll keep the community updated with our latest data. If you’re a researcher or service provider that has a unique NGS application, we’d like to hear about it ! For inquiries or suggestions please contact us at support@genohub.com.

ASHG 2013 Poster Buzz

This year’s ASHG 2013 meeting did not disappoint. As expected, while most talks weighed heavily on understanding genome variation, there was certainly a trend in discussion related to the transcriptome. This was nicely summarized by Tuuli Lappalainen, “almost nothing in the genome makes sense, except in the light of the transcriptome.” While the plenary and platform talks were excellent, I’ve found the poster sessions to be the most valuable. They give you an opportunity to really understand the basis for a particular study and usually talk with the person who has designed the experiment or actually held the pipette (or keyboard). This year there were 3,095 posters. While sitting in my hotel room sifting through the abstracts I wanted to attend, I hit Ctrl-F for a few keywords and the results were interesting. There were several graphical depictions of Twitter buzz during the whole meeting and specifically the plenary talks, below are examples of ASHG poster abstract buzz:

Platforms:

ASHG Poster Sequencing Sequencing Platform Mentions

Library Preparation Types:

barchart_libprep_mentions

Other Keywords:

As an intelligent marketplace that connects researchers with NGS sequencing service providers, understanding what researchers are looking for when choosing services is a critical component to our business. Having the opportunity to attend the poster sessions at ASHG and learn about the latest eQTL and epigenetic changes, GWAS or SNV study keeps us up to date with the latest genome research, ensuring our consultation services on Genohub stays fresh.  Thanks to all those poster presenters who spent time with us at ASHG!