Amplicon Sequencing – Short vs. Long Reads

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

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/ .

Ribosome Profiling (Ribo-Seq): A High-Precision Tool to Quantify mRNA Translation

RNA-Seq has been used consistently for years as a way to determine gene expression by correlating mRNA levels to protein levels. However, the actual translation process in vivo cannot be completely captured by this method. This is because each mRNA molecule isn’t necessarily translated into protein by ribosomes. Ribosome Profiling was developed to help complete this picture.

In this blog, we’ll go over what Ribosome Profiling is, some real-world applications, a typical workflow, and how Genohub can help you with your Ribo-Seq project.

What is Ribosome Profiling?

In order to synthesize proteins, cells transcribe mRNA from DNA and then translate proteins from mRNA. Many researchers who want to study this gene expression process have used RNA-Seq, which provides data on the relative levels of mRNA within a cell. While the levels of specific mRNA often do correlate with the levels of particular proteins, standard RNA-Seq cannot provide actual data regarding gene regulation at the translational level. This is where Ribosome Profiling (Ribo-Seq) comes in.

Ribo-Seq is a sequencing method that uses specific ribosome-protected mRNA fragments (RPFs) to determine the mRNAs that are actively being translated in vivo. This snapshot can then be compared to parallel RNA-Seq done for the transcriptome to reveal the positions and amounts of ribosomes on any specific mRNA.

What are the applications of Ribo-Seq?

Ribo-Seq can help identify alternative mRNA translation start sites, confirm annotated open reading frames (ORFs) and upstream ORFs that may be involved in translation regulation, the distribution of ribosomes on an mRNA and the rate at which ribosomes decode codons. As Ribo-Seq can provide data about gene expression, protein synthesis and protein abundance, it can be useful in almost every type of research, including research on cancer, autoimmune disease, heart disease, neurological disorders, and psychiatric disorders.

The following are examples where Ribo-Seq was used in different types of research.

  • Scheckel et al. used Ribo-Seq in combination with another technique to discover that aberrant translation within the glia only may be enough to cause severe neurological symptoms and may be a primary driver of prion disease.
  • In this paper, the authors summarize multiple studies where Ribo-Seq was used to identify novel genes within plants that could be useful to increase yield through biotic and abiotic stress tolerance if manipulated.
  • In this article, Ribo-Seq was used to reveal translated sequences within long noncoding RNAs and to identify other micropeptides within two herpesviruses, human cytomegalovirus and Kaposi’s sarcoma-associated herpesvirus. Understanding viral gene regulation and other aspects of the proteome are important for understanding their life cycle and identifying epitopes they may present for immune surveillance.

What is the typical Ribo-Seq workflow?

The typical Ribo-Seq workflow begins with collecting and preparing the lysate. First, the cells or tissue samples are harvested and flash-frozen to halt translation. Then, the samples are resuspended in a lysis buffer that includes a salt to stabilize the ribosomes, detergent to puncture the cell membrane, a deoxyribonuclease to degrade genomic DNA, a translation-inhibiting drug to halt the ribosome, and a reducing agent to stop oxidative compounds from interfering with RNA. After lysis, ribonucleases are added to digest the RNA that is not protected inside of the ribosomes. These fragments are called RNA protected fragments (RPFs). Then size selection is performed to identify the ~28 nucleotide RPFs on a gel, and RNA extraction is extracted. Any contaminating rRNA is removed, the RPFs are reverse-transcribed to cDNA, amplified by PCR and then made into libraries that are sequenced.

The data analysis done will ultimately depend on the researcher’s personal aim, but in general, ribosome profiling mapping would include data QC, demultiplexing and then removal of adapter sequences and any remaining rRNA contaminants. The samples would then be aligned to an annotated genome/transcriptome and then counts of the number of reads aligned to each gene would be obtained. These mapped RPFs can then be visualized and compared with what other researchers have done. More specific analysis can include uORF detection, differential gene expression, global translation rates, ribosome stalling, and codon decoding rates.

Where can I get help with my Ribo-Seq project?

As of now the Illumina kit for Ribo-Seq, TruSeq Ribo Profile or ART-Seq, has been discontinued. There is a commercially available all-inclusive library preparation kit, called LACESeq by IMMAGINA Biotechnology. However, Ribo-Seq sample and library preparation is so complex and sample-specific that many labs have their own protocols optimized for their specific samples and then use their favorite commercial small RNA-Seq kit for the last part of library prep. For labs that don’t focus on this type of work, optimizing such a protocol can be very time-intensive and expensive.

Genohub’s Ribo-Seq partners are experts in every step of the Ribo-Seq process, from lysis to custom data analysis, including preparing and running RNA-Seq libraries in parallel, allowing for the measurement of translation efficiency. Our in-network partners also have experience in isolating ribosome-bound mRNA from many different types of samples, including bacteria and eukaryotic cells, and animal and plant tissue. Their proprietary optimized Ribo-Seq protocols means they routinely produce high-quality libraries efficiently and effectively. All you would have to do is provide your frozen cell or tissue samples and let us do the rest.We will be with you every step of the way, from extraction to data analysis! Get started today by letting us know about your Ribo-Seq project here: https://genohub.com/ngs/ .

Illumina Unveils NextSeq 1000 & NextSeq 2000

Last week at the J.P. Morgan Healthcare Conference, Illumina presented their new sequencers, the NextSeq 1000 and NextSeq 2000. 

Strengths: The NextSeq 1000 and 2000 use patterned flow cells similar to the NovaSeq 6000 System that offer the highest cluster density flow cell of any on-market NGS system. To take full advantage of these higher density flow cells, they feature a novel super resolution optics system that is optimized to increase cluster brightness, reduce channel cross-talk, and improve signal-to-noise ratio. This should increase the output and reduce the cost per run compared to the previous NextSeq model (1). The system uses fluors, which both excite and emit with blue and green wavelengths. 

The major difference between the NextSeq 1000 and 2000 capacities is that only the 2000 will be able to handle the larger P3 flowcell. To compare the P2 and P3 flowcells at the 2×150 read length, the P2 flowcell will yield a similar number of clusters to the NextSeq 550 Hi Ouptut kit for a similar runtime. The P3 flowcell will yield a number of clusters that is between the NovaSeq’s SP and S1 flowcells, although the run time is longer, which is likely due to the new super resolution technology. According to Illumina, the NextSeq 2000 will have a $20 per Gb cost, and the NextSeq 1000 will have a $30 per Gb cost (2). 

Regarding downstream data analysis, these new sequencers also come with the DRAGEN system, which is both on-board and cloud-based. The DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT Platform will enable our providers to automate a variety of genomic analysis, including BCL conversion, mapping, alignment, sorting, duplicate marking, and variant calling. According to Illumina, results can be generated in as little as 2 hours (1).

On the wet bench side of things, the NextSeq 1000 and 2000 reagents will also reduce the volume of the sequencing reactions. This volume reduction should decrease waste and minimize physical storage requirements. For example, one cartridge includes all reagents, fluidics and the waste holder (1), which will simplify library loading and instrument use. This should increase efficiency, reduce the chance of user error, lower the sequencing costs, improve recyclability and minimize waste volume. Ideally, these cost savings will then be passed on to our clients. 

Applications: According to Illumina, the new applications available on the NextSeq 1000 and 2000 are small whole-genome sequencing, whole exome sequencing and single-cell RNA-Seq (1), applications which are useful for research in oncology, genetic disease, reproductive health, agrigenomics, etc. 

As some analysis examples, the new DRAGEN Enrichment Pipeline can be applied to whole exome sequencing and targeted resequencing with alignment, small variant calling, somatic variant calling, SV/CNV calling and custom manifest files. The DRAGEN RNA Pipeline can be applied to whole transcriptome gene expression and gene fusion detection with alignment, fusion detection and gene expression. Other standardized DRAGEN pipelines include DRAGEN-GATK, DNA/RNA targeted panels and single-cell sequencing. A more complete list is available here.

Release Date: The NextSeq 2000 is available for order now, but both the NextSeq 2000 and 1000 will only be available for shipment in Q4 2020. The NextSeq 1000 has a list price of $210,000 and the NextSeq 2000 has a list price of $335,000 (2). We have already added the instrument specifications to our database, so providers can start listing their NextSeq 1000 and 2000 services as soon as they are ready.  

Overall, the new NextSeq 1000 and 2000 seem like solid desktop upgrades and also good testing ground for the new super resolution technology. If it goes well, there may be an upgraded version of the NovaSeq unveiling in the future.