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Illumina’s MiSeq is quickly becoming the sequencer of choice for researchers, such as yourself, to carryout their genomic and targeted resequencing studies. The highly experienced team at MR DNA is…
The NovaSeq 6000 System is the latest high throughput sequencing instrument released by Illumina. With up to 6Tb of data and 20 Billion paired-end reads made possible by the Illumina NovaSeq...
The PacBio Sequel is the newest sequencing platform released by Pacific Biosciences. The PacBio Sequel, aptly named, follows the release of the PacBio RS II System; one of the first to offer read lengths greater than 20Kb, and the PacBio Sequel is no different...
The addition of the HiSeq 2500 to our portfolio of sequencers allows us to better serve you, our customers. The innate flexibility of this instrument provides us with the…
Traditional methodology with revolutionized technology. The Applied Biosystems SeqStudio Genetic Analyzer supplies an innovative approach to capillary electrophoresis.
Syntroph diversity and abundance in anaerobic digestion revealed through a comparative core microbiome approach
Anaerobic digestion is an important biotechnology treatment process for conversion of waste to energy. In this study, a comparative core microbiome approach, i.e., determining taxa that are shared in functioning digesters but not shared in non-functioning digesters, was used to determine microbial taxa that could play key roles for effective anaerobic digestion. Anaerobic digester functions were impaired by adding the broad-spectrum antimicrobial triclosan (TCS) or triclocarban (TCC) at different concentrations, and the core microbiomes in both functioning and non-functioning anaerobic digesters were compared. Digesters treated with high (2500 mg/kg) or medium (450 mg/kg) TCS and high (850 mg/kg) TCC concentrations lost their function, i.e., methane production decreased, effluent volatile fatty acid concentrations increased, and pH decreased. Changes in microbial community diversity and compositions were assessed using 16S rRNA gene amplicon sequencing. Microbial richness decreased significantly in non-functioning digesters (p < 0.001). Microbial community compositions in non-functioning digesters significantly differed from those in functioning digesters (p = 0.001, ANOSIM). Microbes identified as potentially key taxa included previously known fatty acid-degrading syntrophs and amino acid-degrading syntrophs. A diverse group of syntrophs detected in this study had low relative abundance in functioning digesters, suggesting the importance of rare microbes in anaerobic digester operation. The comparative microbiome approach used in this study can be applied to other microbial systems where a community-driven biological phenomena can be observed directly.
Fujimoto, M., Carey, D. E., Zitomer, D. H., & McNamara, P. J. (2019). Syntroph diversity and abundance in anaerobic digestion revealed through a comparative core microbiome approach. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-019-09862-4
16s sequencing is one of the go to sequencing methods to complete microbiome studies. 16s sequencing involves sequencing of the 16s rRNA gene found in all bacteria and archaea. The fact that the 16s rRNA gene can be found in all prokaryotes make the 16s gene the ideal candidate to characterize the microbiome of numerous environments. Another key factor that makes the 16s gene an ideal candidate for microbiome characterization is the fact that there are both highly conserved regions and highly variable regions within the gene. The highly conserved regions within the 16s rRNA gene make great targets for PCR primers bind to and replicate thereby producing millions of copies of the targeted hypervariable region that is flanked by the highly conserved regions. The 16s rRNA gene contains 9 hypervariable regions that can be targeted in order to gain insight into the diversity amongst prokaryotes.
16s rRNA sequencing has grown in popularity over the last decade due in large part to next-generation sequencing. NGS, also known as high-throughput sequencing, has drastically reduced the cost of DNA sequencing due to its capability to sequence hundreds of samples at a time. In the past, capillary electrophoresis was able to produce ~1Mb per run, and today, with instruments such as the Illumina HiSeq, we are able to produce ~500 Gb per run. Until recently, one metric that Sanger sequencing had as an advantage over NGS instruments such as the Illumina MiSeq and Ion Torrent was read length. On average, capillary electrophoresis was able to achieve read lengths up to 1,000bp in comparison to the 400-600bp read lengths provided by Illumina and Ion Torrent. However, the PacBio Sequel now allows researchers the ability to achieve read lengths ranging from 10-30Kb. What does this mean for 16s rRNA sequencing? We can now sequence the entire 16s rRNA gene, which is approximately 1.5Kb in length. Whether your goal is to target one specific hypervarible region or the entire 16s rRNA gene, there is a NGS platform for you.
Metagenome sequencing is a term that can cause a lot of confusion, as well as bring so much joy to microbiologists around the world. Metagenome sequencing is a sequencing method that investigates the DNA extracted from an environmental sample as a whole. Whereas certain DNA sequencing techniques will target a specific organism from a specific environment, metagenome sequencing targets all microbial organisms found in a certain environment. There are predominantly two methods used to completely metagenomics studies, 16s rRNA sequencing and Shotgun metagenome sequencing. The difference between the two sequencing methods can be found in their names. 16s rRNA sequencing, as you would expect, targets only the 16s rRNA gene, while Shotgun metagenome sequencing targets all genes present in your sample. How is this accomplished?
Shotgun metagenome sequencing involves the random shearing of all DNA present in a particular sample. These smaller fragments are sequenced on NGS platforms such the Illumina MiSeq and then reassembled. Because shotgun metagenomics is non-discriminatory, not only are you able to gain taxonomic information, similar to 16s sequencing, but you are also able to gain insight to the presence of functional genes as well. Overall, shotgun metagenome sequencing provides a more complete picture of your environmental sample. NGS platforms such as the Illumina HiSeq allow researchers to ability to sequence their metagenome samples to a much greater depth. Why is sequencing depth important? Naturally, the most abundant organisms in your sample will receive the most amounts of data, and because you are not specifically targeting certain organisms, without sufficient sequencing coverage, you run the risk of not identifying those organisms that may be underrepresented. From the human microbiome, to pond water, and to the rumen of cattle, shotgun metagenome sequencing paired with NGS platforms has the ability to take your ecological study to the next level.
The human microbiome continues to be a field of major focus, not just for the scientific community, but for scientists and average joe alike. Whether you're a teenager searching for the next self-help tip to clear up your acne, or your a leading researcher investigating the link between the bacteria in our GI tract and disease such as Alzheimer's or Diabetes, the secrets the human microbiome can provide are becoming all encompassing. Not only is the human microbiome a point of interest for everyone across the world, but is now a point of interest for everyone out of this world. Astronaut Scott Kelly was the first man to spend a full year in space. As if being the first person to spend a year in space didn't make him unique enough, Scott is also a twin. Scott and his brother, Mark, a fellow astronaut, have undergone numerous studies upon Scott's return to Earth. A research group from Northwestern University has undertaken the task of investigating the effects of gravity on the human microbiome. The scientific community is well aware that certain changes to your diet can alter the Bacteroidetes and Firmicutes ratio, but we now know that this ratio is not only altered by our diet but by the force of gravity placed on our bodies. While still early in the study, Fred W. Turek and the rest of the team have found something of significant interest when comparing the microbiomes of the Kelly twins. The bacterial diversity found in Scott remained more constant in comparison to his earth-bound brother. So if you're one of the few who has booked the next rocket ship to space, along side Richard Branson, remember to take a few samples while in space and become a part of not just citizen science, but galactic science.
Source: Fellman, M., (2017). Change in astronaut’s gut bacteria attributed to spaceflight. Northwestern Now. https://news.northwestern.edu/stories/2017/february/change-in-astronauts-gut-bacteria-attributed-to-spaceflight/
Type 1 diabetes, also known as juvenile diabetes, only comprises ~5% of those diagnosed with diabetes. Unlike type 2 diabetes, individuals diagnosed with Type 1 diabetes cannot resort to change in lifestyle or diet with the hopes of no longer being diabetic. Type 1 diabetes is a result of an autoimmune process where cells that should produce insulin are destroyed thereby eliminating that individual from processing sugar into energy. While the onset of Type 1 diabetes cannot be prevented or cured with a change in diet per se, researchers have discovered that the gut microbiota may play a key role in reducing the likelihood of disease occurrence. Stewart et al., have used previous research, documenting the relationship between Type 1 diabetes and the gut microbiota, as a launching point towards investigating the microbiome of individuals diagnosed with Type 1 diabetes with good glycaemic control and a high level of physical fitness in comparison with healthy individuals. The research team successfully characterized the gut microbiota by sampling 20 stool samples and performing 16s rRNA sequencing using the Illumina MiSeq platform. The results of this study indicate that the microbiome of Type 1 diabetics who have optimal glycemic control and good physical fitness closely resembles that found in non-diabetic individuals. Future research may find that, because it is possible to exhibit a diverse and well balanced microbiota under well-maintained Type 1 diabetes, alterations to the gut microbiota may be able to influence the onset of such a disease.
Source: Stewart CJ, Nelson A, Campbell MD, et al. Gut microbiota of Type 1 diabetes patients with good glycaemic control and high physical fitness is similar to people without diabetes: an observational study. Diabet Med. 2017;34(1):127-134.
One of the key areas of metagenomic and microbiome research has been on the transmission of bacteria from mother to infant . Asnicar et al., recently published a study where they sampled 5 mother-infant pairs collecting both fecal and breast milk samples. In order to characterize the microbiome of each pair, DNA was extracted and prepared for metagenomic sequencing on the Illumina HiSeq 2500. Both breast feeding and delivery method have both been seen to play an important role in the diversity of the microbiome of infants relative to their mothers. A vaginal delivery has been documented as providing the infant with a much more diverse microbiome thereby decreasing the likelihood of the infant from succumbing to certain health risks a newborn would experience following a C-section. The research team from Italy postulates that the seemingly apparent transmission of bacteria from mother to infant based on species commonality may not be the most thorough investigative method. "It is therefore imperative to assess whether shared microbes belong to the same genetic variant (i.e., strain) to support the hypothesis of vertical transmission.", states Asnicar et al. Through shotgun metagenomics, the research team was able to assess that based on strain similarity, there does appear to be evidence in support of vertical transmission of the mother's microbiome to their infant. Even as early as 3 months does the microbiome of the infant begin to shift to to what the research team describes as "mother-like".
Source: Studying Vertical Microbiome Transmission from Mothers to Infants by Strain-Level Metagenomic Profiling. mSystems. 2017;2(1):e00164-16.
Have we entered a new era DNA sequencing? An era where DNA sequencing has become so commonplace that the curiosity of the general public is able to inspire new scientific studies. An era of "cultural genomics". The latest study of the Canadian beaver seems to be one of the first endeavors in "cultural genomics." The research team, based in Toronto, states , "We release the beaver genome to mark Canada’s sesquicentennial, and hope the initiative will catalyze other exploratory investigations in “cultural genomics,” of which this project was motivated by a nation’s curiosity and the pride in the animal that has most shaped its history." The world of DNA sequencing and genomics has come a long way since the human genome project. Popularized by "CSI" TV shows and citizen science projects, the general public has become very familiar with the overall concept of DNA sequencing and the capabilities available to the scientific community. As DNA sequencing methods, such as whole genome sequencing, continue to drop in price, the more the scientific community can take an interest in the concerns and ideas of their surrounding communities and investigate topical issues like the history of the Canadian beaver. Lok et al., utilized both the PacBio RSII and Illumina HiSeq X systems to preform de novo genome and de novo transcriptome sequencing. Both of these technologies have allowed the cost of genome and transcriptome sequencing to decrease dramatically and the sequencing power to increase. The results of the genome assembly generated by the Illumina HiSeq X indicate that the research team was able to achieve 160x coverage of the 4 billion bp beaver genome. As sequencing power and popularity continue to progress, it is not difficult to imagine whole genome sequencing becoming a routine test ordered by your family physician.
Source: Lok S, Paton TA, Wang Z, et al. De novo genome and transcriptome assembly of the Canadian beaver (Castor canadensis). G3 (Bethesda). 2017;
In the past few years the world of DNA sequencing has seen a rapid appearance of new next-generation sequencing instruments. Some the new comers include the Illumina NextSeq and the recently announced Illumina NovaSeq. Other companies that are making a niche for themselves in this market are PacBio, with their Sequel system, and Oxford Nanopore, with their MinIon. Ma, Stachler, and Bibbly, recently published a study evaluating the abilities of the Oxford Nanopore MinIon to successfully complete 16s rRNA microbiome studies. With run costs similar to that of the more popular Illumina MiSeq, and a footprint the size of your thumbdrive, it is easy to see what scientists from all over the world are eager to see this "mini" sequencer in action. One of the immediate benefits of the MinIon over some of the more popular NGS platforms is its ability to perform real-time data analysis. But can we have speed without sacrificing accuracy? The team from the University of Pittsburgh found that the data generated by the MinIon did not successfully characherize 6 of the 11 phyla detected by the Illumina Platfrom. Overall, Oxford Nanopore's MinIon is not able to produce the necessary results to successfully capture the microbial diversity in 16s rRNA microbiome studies. The high error rates that are known to plague the MinIon contribute greatly the lack of taxonomic identification. The entire research world is waiting to see if Oxford Nanopore can increase their run quality, because with their run cost and speed, paired with their read length, the MinIon is on the verge of being a vital part of the world of microbiome research.
Source: Ma, X., Stachler, E., & Bibby, K. (2017). Evaluation of Oxford Nanopore MinION Sequencing for 16S rRNA Microbiome Characterization. doi:10.1101/099960
On January 10, 2017, The National Institutes of Health opened an initiative to encourage applicants for funding of genomic research with a special interest in identifying variants in the human genome that may be linked to substance abuse. Previous studies have provided evidence for the existence of a link between the our DNA and the likelihood of an individual developing an addition to habit forming substances. There are ~ 3.5 billion Americans addicted to alcohol, tobacco, and/or illicit drugs, and it is safe to say this initiative set forth by the NIH is a necessary step in improving the public health of this country. With the hopes of identifying genetic risk factors via whole-genome sequencing, this type of federal funding may be just what scientists need to reduce the millions of deaths that result each year stemming from addiction. Researchers such as Wang et al., have focused their efforts on investigating the rare variants related to addiction. In order to identify these rare variants, investigators are utilizing the sequencing power of next-generation sequencing instruments to complete whole-genome and whole-exome sequencing studies. The sequencing depth that NGS instruments such as the Illumina HiSeq provide has uncovered thousands of variants, but unfortunately, the majority of these variants have only been identified from a select group of candidate genes linked to substance abuse. Clearly, based on this funding initiative, this is an area of research the NIH is looking to expand and not limit to only identifying certain variants, but to gain a greater comprehension on gene expression, the effects of small RNAs, and disease progression.
Source: Wang S, Yang Z, Ma JZ, Payne TJ, Li MD. Introduction to deep sequencing and its application to drug addiction research with a focus on rare variants. Mol Neurobiol. 2014;49(1):601-14.
Next-generation sequencing is not only opening doors into the future, but also provides a window into the past. Once limited to only mitochondrial sequencing, DNA sequencing of ancient organisms has excelled leaps and bounds since the early 1980s. This amount of growth in such a short amount of time is due to technological advancements in high-throughput sequencers developed by Illumina, PacBio, and Ion Torrent (Thermo Fisher). From mtDNA sequencing to whole genome sequencing, NGS platforms offer researchers the opportunity to sequencing ancient DNA with much greater specificity and depth than ever before. Devault et al., recently published a study in eLIFE documenting the sequencing of bacteria extracted from 30 year old woman buried 800 years ago near the ancient city of Troy. The research team was able to take advantage of modern DNA extraction kits, such as the MoBio PowerSoil kit, as well as the Illumina MiSeq and HiSeq sequencing platform to not only perform mtDNA sequencing, but whole genome sequencing with up to ~300x coverage. This type of sequencing depth even a few years ago would have been unheard of, but today is common place for both modern day and ancient DNA samples. The two bacterial species sequenced were Staphylococcus saprophyticus and Gardnerella vaginalis. Devault et al. discovered that while G. vaginalis closely resembled that which we would find today, S. saprophyticus more closely resembles that which would be found in the from the time period this woman was buried.
Source: Devault AM, Mortimer TD, Kitchen A, et al. A molecular portrait of maternal sepsis from Byzantine Troy. Elife. 2017;6
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