Tag Archives: High Throughput Sequencing

Amplicon Sequencing – Short vs. Long Reads

Posted on by

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

Posted on by

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/ .

8 Top Online Calculators for Next Gen Sequencing

Posted on by

When it comes to next gen sequencing, here are the top 8 online calculators that we have found:

Coverage

  1. Genohub’s shop by project feature
  2. The Coverage Calculator by Clinical Genomics

Oligonucleotide

  1. The Oligo Calc: Oligonucleotide Properties Calculator by Northwestern University
  2. The OligoAnalyzer 3.1 by Integrated DNA Technologies

Primers

  1. NCBI/ Primer-BLAST: Finding primers specific to your PCR template (using Primer3 and BLAST) 
  2. Primer3: WWW primer tool by UmassMed

qPCR Efficiency

  1. The qPCR efficiency calculator by Thermo Scientific
  2. The QPCR Standard Curve Slope to Efficiency Calculator by Agilent Technologies

If you know of any other great online calculators for next gen sequencing please tell us about it!

BaseClear Lists Next Gen Sequencing Services On Genohub

Posted on by

Genohub would like to welcome BaseClear to the Genohub family. The sequencing services listed by BaseClear has added more available options for researchers on the Genohub next generation sequencing market.

BaseClear has listed the following next gen sequencing instruments and Illumina library prep options:

  • Instruments:
    • Illumina MiSeq paired end sequencing options
    • Illumina HiSeq paired end sequencing options
    • PacBio SMRT Cell
  • Library prep:
    • Illumina DNA
    • Illumina 16S V4
    • Illumina RNA (rRNA-depleted)

BaseClear is located in the BioSciencePark (BSP) of Leiden in the Netherlands. By using Illumina systems (HiSEQ2500 and MiSEQ) in combination with the PacBio RS system BaseClear can offer whole genome analysis and offer a wide variety of combinations of read lengths, number of tags and paired end size ranges. The lab has been ISO17025 accredited since 2006.

We are excited to include BaseClear’s high throughput sequencing services on the Genohub next gen sequencing market.

UTHSCSA Lists Next Generation Sequencing Services On Genohub

Posted on by

Genohub would like to welcome the Genome Sequencing Facility (GSF) at Greehey Children’s Cancer Research Institute in the University of Texas Health Science Center at San Antonio to the Genohub family. The sequencing services listed by UTHSCSA has added more available options for researchers on the Genohub next generation sequencing market.

UTHSCSA listed the following next generation sequencing instruments and Illumina library preparation options:

  • Instruments:
    • Illumina MiSeq paired end sequencing options
    • Illumina HiSeq single read and paired end sequencing options
  • Library prep:
    • Illumina ChIP
    • Illumina Directional RNA (polyA-selected)
    • Illumina Directional RNA (rRNA-depleted)
    • Illumina DNA
    • Illumina Exome
    • Illumina RNA (polyA-selected)
    • Illumina RNA (rRNA-depleted)
    • Illumina Small RNA (microRNA)

The GSF has a stated mission to deploy NGS systems focusing on cancer genomics to benefit Greehey CCRI investigators and the wider research community. The NGS facility performs all protocols necessary for preparing a sample to be sequenced on the Illumina HiSeq 2000 system. The GSF is also a research group with experience in customizing experiments and developing new applications. They provide:

  • Complete project consultation for optimal experimental design and setup
  • Complete quality control analysis during library preparation and data generation
  • Secure website to download the sequencing data with quality information
  • Full project report
  • Bioinformatics analysis option available
  • NCBI GenBank submission option available

We are excited to include UTHSCSA high throughput sequencing services on the Genohub next generation sequencing market.

Micro-Seq Lists Next Generation Sequencing Services On Genohub

Posted on by

Genohub would like to welcome Micro-Seq Enterprises to the Genohub family. The sequencing services listed by Micro-Seq has added more available options for researchers on the Genohub next generation sequencing market.

Micro-Seq listed the following next generation sequencing instrument and Illumina library preparation options:

  • Instrument:
    • Illumina MiSeq paired end sequencing options
  • Library prep:
    • Illumina DNA
    • Illumina 16S V4
    • Illumina Small RNA (microRNA)
    • Illumina RNA (rRNA-depleted)
    • Illumina Bisulfite

Micro-Seq Enterprises was formed to help reduce the sometimes long delay researchers are forced to endure to get the results of their experiments that involve DNA or RNA sequencing (RNA-seq, RNAseq), including bacterial community analysis (16S sequencing). Micro-Seq is able to offer quick turnaround times for researchers with smaller sequencing projects. Unlike sequencers with enormous capacity, the economic scale of the Illumina MiSeq prevents researchers with smaller sequencing projects from enduring longer wait times and higher fees.

 

We are excited to include Micro-Seq’s high throughput sequencing services on the Genohub next generation sequencing market.

Press Release: Genohub Launches Next Generation Sequencing Marketplace

Posted on by

You can view the original press release on our official launch at PRWeb.com. Thanks to the folks at RNA-Seq Blog for quickly picking up the story.

Genohub announced the launch of their online market for next generation sequencing services today.

Austin, TX (PRWEB) August 16, 2013

Genohub.com announced the launch of their online market for next generation sequencing services today. The online service, is positioned to completely change the way high throughput sequencing services are ordered, accelerating genomic research by improving access to sequencing services. Genohub’s intelligent sequencing matching engine instantly matches researchers with service providers based on specific project criteria. Genohub facilitates the management of sequencing projects throughout the sequencing lifecycle from selecting orderable sequencing packages, to communication, payments and delivery of data.

For Researchers

Genohub’s online service transforms the way researchers go about ordering next generation sequencing (NGS) and reinforces the critical researcher-provider communication cycle involved in every project. Genohub’s model eliminates the need for researchers to call multiple service providers to compare service details and prices. Researchers use the smart NGS matching engine to gain immediate access to up-to-date service listings from reputable providers. The transparent pricing model, with exact service prices, reduces the time needed to compare services and makes it significantly faster, more informed and more accurate than manually ordering by email or phone. Researchers using the service are also able to take advantage of one-time deals and other offers not normally available through a provider’s website or pricing sheet. Clear maximum turnaround times for each service reduces the unpredictability associated with project completion dates. Researchers using the service are able to track the status of their orders, upload data or project specific information and post messages to providers performing the work.

Genohub’s shopping interface is designed to accommodate both researchers with prior experience with the latest sequencing technology, as well as the increasing number of life science researchers who are not necessarily familiar with the latest sequencer specs or perhaps have no prior sequencing experience at all. Experienced users can search by selecting specific instruments and run types while researchers new to sequencing can shop for services by their project requirements, e.g., read number and coverage. Researchers who need help selecting the right sequencing service can also take advantage of free consultation by Genohub’s PhD trained staff.

For Service Providers

Genohub has also invested significantly on promoting NGS service providers by allowing them to advertise services and extend their reach to places where they would normally not receive orders. Providers sign up and list their services in a structured format allowing Genohub’s matching engine to automatically offer services to customers based on their experimental needs. The online service facilitates customer communication via a centralized messaging interface, which allows providers to request data, convey unforeseen handling or quality issues and relay project status to the researcher.

Genohub also automatically generates accurate quotes based on the pricing information that has to be entered by the provider only once. This significantly reduces the amount of time providers would normally have to spend on creating and communicating quotes

“While high-throughput sequencing holds enormous potential for unlocking new discoveries, the high cost and complexity of sequencing projects necessitate a professional marketplace like Genohub to improve access and facilitate collaboration between researchers and service providers across universities, companies, as well as other private and public research organizations around the globe,” said Pouya Razavi, Genohub’s CEO and co-founder.

Media Contact:
Estevan McCalley, Head of Customer Development
Genohub
512-436-0111
info@genohub.com
https://genohub.com/

Global Biologics Lists Next Generation Sequencing Services On Genohub

Posted on by

Genohub would like to welcome Global Biologics to the Genohub family. The sequencing services listed by Global Biologics has added more available options for researchers on the Genohub next generation sequencing market.

Global Biologics listed the following next generation sequencing instrument and Illumina library preparation options:

  • Instrument:
    • Illumina HiSeq paired end sequencing and single read sequencing options
  • Library prep:
    • Illumina DNA
    • Illumina DNA Hydroxymethylation
    • Illumina ChIP
    • Illumina Small RNA (microRNA)
    • Illumina Directional RNA (polyA-selected)
    • Illumina Bisulfite
    • Illumina Mate Pair (7-15 kb)

Global Biologics is a research & development service laboratory focused on clients performing discovery and applied research from academic, government, & private organizations. With experienced personnel and efficient molecular services, Global Biologics can aid scientists in advancing complex research studies.

The nucleic acid services offered by Global Biologics address common laboratory bottlenecks which are critical yet time consuming to perform. Specifically, Global Biologics has expertise in the following areas:

  • NGS Library construction & Sequencing (Illumina)
  • PCR based gene expression profiling and genotyping
  • Targeted Sequencing Applications (HiSeq) & Data Analysis
  • Nucleic Acid Isolation, QC and archive
  • Automation, scale up, and tech transfer

We are excited to include Global Biologic’s high throughput sequencing services on the Genohub instant matching engine.

The Institute for Personalized and Predictive Medicine of Cancer (IMPPC) Lists High Throughput Sequencing Services On the Genohub Market

Posted on by

We want to welcome The Institute for Personalized and Predictive Medicine of Cancer (IMPPC) to the Genohub family. The high throughput sequencing services listed by IMPPC will add more available options to the Genohub next generation sequencing market.

IMPCC listed the following next generation sequencing instruments and library prep options:

  • Instruments:
    • Illumina HiScanSQ
    • Ion PGM 314 Chip
    • Ion PGM 316 Chip
    • Ion PGM 318 Chip
  • Library prep:
    • Illumina Directional RNA (polyA-selected)
    • Illumina Small RNA (microRNA)
    • Ion DNA

The IMPCC is a publicly funded institute backed by a private foundation with a mission to advance in the understanding of genetic and epigenetic factors leading to higher predisposition or susceptibility to develop cancer. We look forward to matching IMPPC with the most compatible high throughput sequencing projects for their lab.

Axeq Technologies Lists High Throughput Sequencing Services on the Genohub Market

Posted on by

We want to welcome Axeq Technologies to the Genohub family. Axeq has listed high throughput sequencing services if offers on the Genohub next generation sequencing market. Axeq currently has 4 worldwide sites:

  • HQ – Axeq USA in Maryland
  • Axeq Europe in Amsterdam
  • Axeq Asia in Seoul
  • Axeq Japan in Tokyo

Macrogen is the parent company of Axeq which has been a phenomenal success in both the scientific services market and in the field of original genetic research. With devotion to molecular biology, cell biology, genetics research, and extensive investment in installations of cutting-edge research tools – including genotyping, Sanger sequencing and next-generation sequencing platforms, Macrogen successfully published numerous articles in leading journals. These include the publication of the first whole genome sequence of a Korean (Nature), and high resolution CNV analysis of the Asian population (Nature Genetics).

Axeq Technologies was formed in early 2011 as a premium brand to offer both scientific services and collaborations to researchers around the world. The company is dedicated to providing a whole new level of service experience based on its ability to provide publication-quality data and 24/7 client support. Currently, Axeq has listed high throughput sequencing services on an Illumina GAIIx instrument. We look forward to matching Axeq with sequencing projects on the Genohub next generation sequencing market.