Tag Archives: paired end sequencing

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/ .

BaseClear Lists Next Gen Sequencing Services On Genohub

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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

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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.

How to Select the Best Next Generation Sequencing Platform For Your Project

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We often get questions from researchers on selecting the best next generation sequencing platforms for specific projects. We are glad to offer free consultation to researchers on inquiries such as these among others. We also offer a sequencing guide to get you started.

Another great resource is a YouTube talk by Dr Elaine Mardis, a professor of Genetics and Molecular Biology and the co-director of the Genome Institute at the Washington University School of Medicine. She begins by discussing next gen sequencing instrument similarities, such as library amplification, nucleotide detection, read lengths, and paired end sequencing. She then dives into the factors unique to individual next gen sequencing instruments by Roche 454, Life Technologies, Illumina, Ion Torrent, PacBio, and Oxford Nanopore. The discussion focuses on the benefits and dissimilarities among them associated with:

  • Paired end reads: linear vs circularized fragments
  • Sequencing technologies: DNA polymerase, DNA ligase, synthesis H+ detection, syntheses, and nanopore
  • Library amplification methods: emPCR, bridge amplification, and its absence in some 3rd gen platforms
  • Run times
  • Error rates
  • Read lengths

Below, you can view the video in its entirety which goes into quite some detail and discusses the best types of projects for each NGS platform.

Micro-Seq Lists Next Generation Sequencing Services On Genohub

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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.