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What is Shotgun Metagenome Sequencing?
What is Shotgun Metagenome Sequencing? How is shotgun metagenome sequencing different from 16s metagenome sequencing? These are valid questions and questions that can be resolved with a quick breakdown of metagenome sequencing methods.

What is the difference between Metagenomics and Metagenome Sequencing?

Metagenomics is the study of microbial populations sampled directly from the environment such as soil from crop fields, pond water, an open wound etc. Metagenomic studies can be completed with a variety of metagenome sequencing methods including:
- Shotgun metagenome sequencing
- 16s rRNA sequencing
- Whole-genome sequencing
- Transcriptome sequencing
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What is 16s Sequencing?
16s rRNA sequencing has become one of the leading methods for phylogenetic studies. The popularization of 16s sequencing methods has been due in large part to the wide availability of PCR and Next-generation sequencing facilities, such as MRDNA. But what is 16s rRNA sequencing? And why should you choose 16s sequencing methods over other DNA sequencing methods?

16s rRNA sequencing refers to sequencing the 16s rRNA gene that codes for the small subunit (SSU) of the ribosome found in prokaryotes such as Bacteria and Archaea. There are several factors that make the 16s rRNA gene the perfect target to complete your taxonomy or phylogeny studies.
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What is the Microbiome?
As DNA sequencing methods continue to develop and continue to become more powerful, scientists are unleashing that power in their research on the interactions of the human microbiota and its effects on human health and development. But what is the microbiome?... or is it microbiota?

First, it is important to distinguish between these two terms: microbiome vs microbiota. The microbiome is characterized as the genomes of the bacteria, archaea, and fungi collectively that reside in a specific environment such as the human body. The microbiota is characterized as the collection of the bacteria, archaea, and fungi themselves. So, using the power of next-generation sequencing, we can gain a greater insight into the human microbiota by investigating the microbiome through metagenome sequencing methods.
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How Much DNA Is Needed for Genome Sequencing?
How much DNA do I need??!! This is one of the most common questions that we receive, and the answer often times is...it depends. As you can expect, the amount of DNA needed is dictated by sequencing instrument and project type. If you are interested in genome or metagenome sequencing on any of the Illumina sequencers such as the Illumina MiSeq or Illumina NovaSeq, the recommended amount of DNA is 50 ng-500 ng. If the genome you are trying to sequence is large or complex, we strongly recommend submitting at least 100 ng of good quality gDNA. Good quality DNA will be free of EDTA, organic contaminants, such as phenol and ethanol, and other inhibitors that may interfere with successful library preparation or DNA sequencing.

But What if I dont't have 50ng of gDNA??? There is no need to worry, there are low DNA input options!!! For small microbial genomes, we can accept as little as 1 ng of gDNA. It is important to note that there will be some optimization required for low-input samples which may increase cost. So, while low-input genome sequencing is possible, we do recommend multiple gDNA extractions if the first attempt did not yield as much DNA as originally expected.
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How Much RNA Is Needed for Transcriptome Sequencing?
How much RNA do I need??!! Well, much like DNA, it depends. Before you can determine how much RNA is needed, the first question that needs to be answered is, "What method of RNAseq are you interested in?" Speaking in general terms, RNAseq or transcriptome sequencing will refer to Total RNA Sequencing, but there are other library prep options other than Total RNA Sequencing such as ribosomal RNA depleted Transcriptome sequencing and poly(A) enriched Transcriptome sequencing. RNA input requirements will also be dictated by sample type e.g. eukaryote RNA vs prokaryote RNA sequencing. Each of these variables will determine how much RNA is needed for transcriptome sequencing.
- Eukaryote RNAseq
  - Sample Type -- Total RNA
  - Quantity -- 1-2 ug (1ug of total RNA approximately equates 50-100ng mRNA after Poly-A selection)
  - Volume -- 25 ul
  - Concentration -- 100 ng/ul
  - RNA integrity number (RIN) -- 8
- Prokaryote RNAseq
  - Sample Type -- Total RNA
  - Quantity -- 100-300 ng
  - Volume -- 25 ul
  - Concentration -- 10 ng/ul
  - RNA integrity number (RIN) -- 8
- Low input RNA
  - Sample Type -- Total RNA
  - Quantity -- 100-300 ng
  - Volume -- 25 ul
  - Concentration -- 10 ng/ul
  - RNA integrity number (RIN) -- 8
- FFPE
  - Sample Type -- Total RNA
  - Quantity -- 200-300 ng
  - Volume -- 25 ul
  - Concentration -- 20 ng/ul
  - RNA integrity number (RIN) -- 8
- rRNA Depletion
  - Sample Type -- Total RNA
  - Quantity -- 1-2 ug*
  - Volume -- 25 ul
  - Concentration -- 100 ng/ul
  - RNA integrity number (RIN) -- 8
Why Are There So Many Contigs?
While genome sequencing has gotten relatively inexpensive over the last several years, genome assembly on the other hand is still rather greedy. What do we mean by greedy? Well, often times a complete genome assembly requires a lot of data to help fill in the gaps. In a perfect world, 1 Genome Assembly would result in 1 Contig on a consistent basis, but unfortunately, we just aren’t there yet (but we are getting close). This is especially true for De Novo Genome Assembly. Generating a single contig from a De Novo Genome Assembly is incredibly difficult due to a number of factors including high repetitive regions which can span thousands of base pairs. One of the key advantages of NGS technology, such as the Illumina MiSeq or Novaseq, over Sanger sequencing is the reduced cost, but one of the key disadvantages is the reduced read length. Because we are often handling reads 150-250bp in length, determining where in the genome these short repetitive regions overlap in order to generate an accurate map of the full-length repetitive region is quite challenging. Unfortunately, this challenge can leave researchers with a lot of gaps to fill…literally. Additionally, the complexity of genome assembly only increases when dealing with polyploid genomes and determining which alleles should be mapped to which loci.

How do I fill in the Gaps?
One of the best ways to fill in the gaps is ...

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Can I Submit Raw Sample? How Much Should I Send?
Of course!! MR DNA is glad to offer our customers DNA and RNA Extraction services for almost any sample type e.g. soil, plant matter, feces, etc. Just name it and there is a good chance we have already developed a successful DNA and/or RNA isolation procedure for downstream next-generation sequencing (NGS). This is one of the key advantages of sending MR DNA your raw samples. We know that there are a variety of purification techniques out there, but unfortunately, not all nucleotide purification techniques were made equally. Over the years, MR DNA has had the exciting opportunity to develop extraction methods for a wide range of sample types, and over these years we have made the necessary adjustments to ensure the yield and purity meet the requirements for NGS library prep and sequencing. By allowing MR DNA to take on the task of DNA/RNA isolation, you may also be saving yourself time and money. Aside from the time saved of nucleotide extraction itself, if the DNA or RNA do not meet the necessary quantities or purity requirements for your desired sequencing method then additional services may be required such as linear amplification or additional rounds of column purification to move forward with your project, and thereby increase the time and cost required.
How Much Sample Should I Send?

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Which Sequencing Platform Should I Choose?
At MRDNA, we pride ourselves on offering our customers the ability to choose from the latest NGS platforms the industry has to offer. But sometimes, having too many options can be confusing...where should you begin? Well, as with most decisions, cost is going to be a significant factor.
Ion S5 XL:
Our least expensive option is going to be the Ion S5 XL. The Ion S5 (aka PGM Sequencing Technology) is the ideal choice for probative studies...for those investigators that aren't too sure what to expect, but are curious to know what/who and how many are in their sample. The limiting factor for the Ion S5 is read length (~300-400bp). Our two most requested assays for the Ion S5 are the 515F-806R amplicon primer pair, which targets the 16s rRNA v4 region, and ITS1-ITS2 amplicon primer pair, which targets a portion of the internal transcribed spacer (ITS) region commonly used for fungal studies. Pricing for these two assays is as low as $60/assay.

Illumina:
The Illumina sequencing platforms are by far the most popular and arguably the most cited NGS technology. The Illumina MiSeq is able to offer read lengths up to 600bp and the Illumina NovaSeq is able to offer read lengths up to 500bp. Of course, smaller read lengths are available depending on your needs. If you are interested in targeted sequencing, but maybe have an interest in regions larger than the 16s v4 region e.g. V1-3, V3-V4, or V5-V7, the Illumina MiSeq is going to the platform for you. Pricing for targeted (amplicon) sequencing for the Illumina MiSeq begins at $85/assay but can be as low as...

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