Tag Archives: NGS library prep

Amplicon Sequencing – Short vs. Long Reads

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Amplicon sequencing is a type of targeted sequencing that can be used for various purposes. Some common types of amplicon sequencing are 16S and ITS sequencing, which are used in phylogeny and taxonomy studies for the identification of bacteria and fungi, respectively. When there is a need to explore the genome more generally, amplicon sequencing can be used to discover rare somatic mutations, detect and characterize variants, and identify germline single nucleotide polymorphisms (SNPs), insertions/deletions (INDELs), and known fusions [1, 2]. Targeted gene sequencing panel projects are another example of amplicon sequencing, where these panels include genes that are often associated with a certain disease or phenotype-of-interest [3].

In this article, we will go over what amplicon sequencing is, describe the advantages and disadvantages of short- and long-read sequencing, and then explain how Genohub can help support your project.

Amplicon Sequencing

Amplicon sequencing is targeted sequencing that involves specific primer design in order to achieve high on-target rates. It’s called amplicon sequencing, because a crucial step of the process is polymerase chain reaction (PCR), which is a method that amplifies specific DNA sequences based on the primers used. Primers are small DNA oligos that are specifically designed to target only the genes/regions-of-interest. When the amplification part of PCR occurs, only these specific genes are multiplied. The final products of PCR are called amplicons, hence amplicon sequencing [1].

It’s important to think about what type of sequencing (short vs. long read) needs to be done for your specific project, because in order to sequence amplicon samples, the appropriate adapters need to be added to help them adhere to sequencing flow cells [2]. These adapters will differ depending on the flow cell, and in some cases, it may even be more cost-effective to send DNA samples and have one of our NGS partners perform all the library prep themselves.

Short read sequencing (Illumina)

Short-read amplicon sequencing is done with Illumina platforms, often the MiSeq, and has been the standard for 16S, ITS and other microbial profiling projects for many years. Being the standard for so long has advantages, as there are many targeted gene panels created and validated already for use with Illumina sequencing, which can make the workflow much easier on researchers who are new to targeted sequencing. There is also an abundance of literature with Illumina sequencing, so it’s easy for researchers to compare their findings to those of other groups. The biggest advantage is that researchers can sequence hundreds of genes in a single run, which lowers sequencing costs and turnaround time, especially if the researcher is interested in many different genes [1].

A disadvantage with short-read sequencing is that the sequencing resolution may not be as high as long-read sequencing. A comparison of short-read to long-read 16S amplicon sequencing showed that only long-read sequencing could provide strain-level community resolution and insight into novel taxa. Then for the metagenomics portion, a greater number of and more complete bacterial metagenome-assembled genomes (MAGs) were recovered from the data generated from long reads [4].

Long read sequencing (PacBio and Nanopore)

Long-read amplicon sequencing is done with either the PacBio or Oxford Nanopore platforms. They both offer complete, contiguous, uniform, and non-biased coverage across long amplicons up to 10 kb. Advantages of this type of long-read amplicon sequencing is that it’s more efficient, accurate and sensitive than short-read sequencing.

PacBio sequencing can obtain up to 99.999% single-molecule base calling accuracy and has been used to sequence full-length 16S and ITS sequences with very high accuracy as well [3].

Nanopore sequencing can provide accurate variant calling as well as robust coverage of larger targeted regions, which can help enhance the analysis of repetitive regions and improve taxonomic assignment [5]. Nanopore sequencing also tends to allow a bit more flexibility than PacBio sequencing when it comes to scaling amplicon projects at a cost-effective price [6].

The disadvantages to using long-read sequencing for amplicon projects is that it tends to be much more expensive and time-consuming than short-read sequencing, and sometimes long reads may not even be needed if the targeted amplicons themselves are already very short.

How can Genohub help you?

Genohub’s amplicon sequencing partners are experts in every step of the amplicon sequencing process, including extraction, PCR amplification, adapter ligation, library prep and data analysis. Our partners have experience extracting from many different types of environmental and biological samples, but they can work just as well with your DNA or amplicons if you prefer to extract and/or perform PCR in your own lab. From our experience, it’s more cost-effective to send DNA samples rather than amplicons, unless you can attach Illumina adapters yourself.

We know that each research project is unique, so we have partners who are also open to working with your custom primers, custom gene panels and custom bioinformatics needs! Get started today by letting us know about your amplicon sequencing project here: https://genohub.com/ngs/ .

Fungal Sequencing – ITS vs. 18S

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Studying the Fungi kingdom is important, because they have so many different ecological roles, including decomposers, symbiotes and parasites. There are also more than 1 million different species of fungi, so researchers need to have high-throughput methods to explore this diversity [1]. One such method is next-generation sequencing.

In this blog, we’ll go over why and how researchers sequence for fungi, what the ITS and 18S genes are, how to choose between them and how Genohub can help with your fungal sequencing project.

Why perform sequencing for fungal community analysis?

Fungal sequencing can be used to discover novel fungal species, quantify known fungi, explore the structure of fungal communities, and determine the roles of fungi in nature. In addition, it’s important to study these communities for human health, as there are some fungi that are resistant to antifungal drugs and others that are involved in plant diseases [2]. Thus, sequencing for fungi is relevant for multiple fields, including environmental conservation, agriculture, and microbiology.

Both ITS and 18S sequencing are well-established methods for studying fungal communities, as focusing on these genes is a simple way to identify fungi within complex microbiomes or environments that would otherwise be difficult to study [3]. For example, this type of specific amplicon sequencing enables the analysis of the fungal community within very mixed environmental samples, such as soil or water.

What are ITS and 18S?

The internal transcribed spacer (ITS) region and the 18S ribosomal RNA gene are used as biomarkers to classify fungi.

Figure 1. Picture of the ITS region as spacers between the ribosomal subunit sequences.

As seen in Figure 1, the ITS region includes ITS1 and ITS2, the spacer genes located between the small-subunit rRNA and large-subunit rRNA. Generally, the ITS1/ITS4 primers are used for amplification of the ITS region, although they can be substituted with the universal primers ITS2, ITS3, and ITS5 [4].

The 18S ribosomal RNA (18S rRNA) gene codes for a component of the small 40S eukaryotic ribosomal subunit and has both conserved and variable regions. The conserved regions can reveal the family relationship among species, whereas the variable regions will show the disparities in their sequences. Regarding the variable regions, 18S rRNA gene has a total of nine, V1-V9. The regions V2, V4 and V9 together are useful for identifying samples at both the family and order levels, while V9 seems to have a higher resolution at the genus level [5].

How to choose between ITS and 18S?

Although both ITS and 18S rRNA have proven useful for assessing fungal diversity in environmental samples, there are enough differences between them that researchers may choose to focus on only one, although sequencing for both is an option as well.

There was relatively low evolutionary pressure for the ITS1 and ITS2 sequences to remain conserved, so the ITS region tends to be hypervariable between fungal species while remaining moderately unchanged among individuals from the same species. It is therefore very well suited as a marker for species identification in the classification of fungus and is often used to study relative abundance of fungi as well [2]. This can be useful if you need to perform a survey for genetic diversity at the species level or even within a species.

On the other hand, there was significant evolutionary pressure for the 18S rRNA gene to remain highly conserved as a component of the small eukaryotic 40S ribosomal subunit, an essential part of all eukaryotic cells. Due to this pressure, 18S is considered a potential biomarker for fungi classification above the species level and is often used in wide phylogenetic analyses and environmental biodiversity screenings [5].

In summary, the ITS region is mainly used for fungal diversity studies, while 18S rRNA is mainly used for high resolution taxonomic studies of fungi.

How can Genohub help?

Genohub’s ITS and 18S sequencing partners are experts in every step of the amplicon sequencing process, including extraction, PCR amplification and library preparation using validated primers based on the literature, and data analysis, including taxonomic assignment, diversity and richness analysis, comparative analysis, and evolutionary analysis. Our partners have experience extracting from many different types of environmental and biological samples, including soil, water, sludge, feces, and plant and animal tissue, but they can work just as well with DNA samples that you extract yourself.

We know that each research project is unique, so we have partners who are also open to working with your custom primers or your custom analysis needs! Get started today by letting us know about your ITS or 18S sequencing project here: https://genohub.com/ngs/ .

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 Next Generation Sequencing Applications

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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

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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!