Tag: Lab Work

Paper published on “Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease”

Sue IshaqLeave a comment

The Ishaq and Li labs at UMaine are delighted to announce that our paper on “Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease.” has been published in mSystems!! ASM was kind enough to write a press release about study, found here.

The complete author list, Abstract, and Ackowledgements/Funders portions of the paper can be found at the end of this post. This paper is part of a larger Broccoli project, in which we are evaluating the use of broccoli sprouts in the diet to enlist gut microbes to produce anti-inflammatories as a way to resolve symptoms of Inflammatory Bowel Disease.

The Premise

Broccoli sprouts are very high in a compound called glucoraphanin, which is in-active for humans. When glucoraphanin comes in contact with the myrosinase enzyme, also found in the sprouts, it is transformed into sulforaphane, which drives away insect pests but acts as an anti-inflammatory in people!

If you eat raw sprouts, most of this conversion happens when you cut or chew the sprouts, and that anti-inflammatory will get absorbed in your stomach. If you steam or cook the sprouts, you can inactivate the enzyme and leave the glucoraphanin compound alone. Some of your gut microbes are able to use glucoraphanin, and produce the anti-inflammatory sulforaphane right in your gut! We are trying to understand how and when this works, so we can use it to reduce symptoms of Inflammatory Bowel Disease.

A diagram with two panels, and a cartoon mouse in the middle. The cartoon mouse is eating broccoli, and a cartoon of the digestive tract is overlaid on the mouse's abdomen. Lines emanating from the broccoli point to the left panel, and show the compound glucoraphanin being converted into sulforaphane by the myrosinase enzyme. Lines emanating from the colon of the mouse point to the panel on the right, showing the same biochemical conversion by gut microbes.
A cartoon of a woman eating broccoli, with the digestive tract shown on her shirt, and smiling microbes in the background.

The mice in this trial are used to mimic Crohn’s Disease, which is one of the main ways that Inflammatory Bowel Diseases may be classified. Crohn’s Disease is complictaed, and involves an over-active immune response to gut microbes. This is replicated in mice that are bred to lack the genes in the DNA to make interleukin-10 (IL-10). IL-10 is an immune factor that can be used to calm the immune system and tolerate microbes which are not causing harm. Without IL-10, these mice over-react to the presence of bacteria, even those which are not causing harm, and this creates symptoms similar to Crohn’s in people.

We used two age groups of mice, and in each group, half ate a mouse chow (control) diet and half ate the mouse chow with 10% of the chow replaced by raw broccoli sprouts. Crohn’s often develops in childhood and adolescence, so our two age groups of mice reflect the juvenile stage (4-5 weeks old) and the adolescence stage (5-6 weeks old) of symptom onset. After wo weeks of symptoms, we sacrificed the mice and collected as much information as we could.

Figure 1 from the paper mentioned in this post. It shows an experimental design.

The Team

The mice, their care during the experiment, and sample collection for this project was graciously provided by University of Vermont researchers Gary Mawe and Brigitte Lavoie, and then-grad-student-now-medical-student Molly Hurd, in 2021. The SUNY Bingamton team, Tao Zhang and Allesandra Stratigakis, processed metabolite and cytokine samples and analyzed those data. The UMaine team (pictured below and led by Sue Ishaq and Yanyan Li) processed and analyzed data from different locations of gut tissue for histolgy and sequencing of bacterial communities, as well as analyzing those data, and took the lead on writing the paper.

Portrait of Lola Holcomb, wearing a block sweater on a beach at sunset
Lola standing in front of her poster which displays the results of her research on gut microbes.
Louisa Colucci. Young woman with dark hair tied in a bun is sitting in a kayak with her paddle raised. She is wearing a black top and a red life-vest over it, in a light blue, single-person kayak. The kayak is in the water, most likely a lake, with the forested shoreline in the background.

The Health Benefits were most obvious in the younger mice

The mice that were eating the broccoli sprouts in their chow and did much better than the control group who ate only mouse chow when symptoms of Crohn’s Disease were induced — and we found something really interesting… The diet worked really well in the younger mice and reduced their symtpoms of inflammation and illness for almost every metric we studied. The older, adolecent mice got some benefit from eating the raw broccoli sprouts, but not nearly as much as the younger mice! Those graphs are shown in the paper.

The Gut Microbes were most changed in the younger mice

Bacterial richness (the number of different types of bacteria present) was increased, but only in younger mice consuming a 10% raw sprout diet, which is useful because pediatric Crohn’s patients usually have fewer types of bacteria present in their gut.

Younger mice consuming broccoli sprouts also had more types of bacteria that are known to convert glucoraphanin into sulforophane, and they had more of the genes needed to do it. Crohn’s patients usually have fewer of these types of bacteria, which are also known to provide other health benefits.

The Next Steps

We are currently working on replicating and expanding this project to include more age groups, so we can understand how different diet preparations of broccoli sprouts impact immune systems and gut microbiota at different developmental periods of life. We are also really interested in understanding how sex in mice, and gender in humans, plays a role in how immune systems and microbial communities develop during a critical phase of life. We have some initial data to suggest that male and female mice respond to different diets and at differnt ages, but we aren’t sure why yet.

We hope to expand our work with people to study how these diets work in the real world, and how we can tailor diet and cooking preparations of sprouts to best meet the needs of people of different ages, health statuses, and tastes.

The paper

Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease. 

Lola Holcomb1$, Johanna M. Holman2$, Molly Hurd3, Brigitte Lavoie3, Louisa Colucci4, Benjamin Hunt5, Timothy Hunt5, Marissa Kinney2, Jahnavi Pathak1, Gary M. Mawe3,Peter L. Moses3,6, Emma Perry7, Allesandra Stratigakis8, Tao Zhang8, Grace Chen9, Suzanne L. Ishaq1*, Yanyan Li1*

1 Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, USA 04469. 2 School of Food and Agriculture, University of Maine, Orono, Maine, USA 04469. 3 Larner College of Medicine, University of Vermont, Burlington, Vermont, USA 05401. 4 Department of Biology, Husson University, Bangor, Maine, USA 04401. 5 Department of Biology, University of Maine, Orono, Maine, USA 04469. 6 Finch Therapeutics, Somerville, Massachusetts, USA 02143. 7 Electron Microscopy Laboratory, University of Maine, Orono, Maine, USA 04469. 8 School of Pharmacy and Pharmaceutical Sciences, SUNY Binghamton University, Johnson City, New York, USA 13790. 9 Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA 48109

$ these authors contributed equally.

Keywords: Crohn’s Disease, cruciferous vegetables, sulforaphane, glucoraphanin, gut microbiota, dietary bioactives, 16S rDNA, interleukin-10 knockout 

Abstract

Crohn’s Disease (CD) is a presentation of Inflammatory Bowel Disease (IBD) that manifests in childhood and adolescence, and involves chronic and severe enterocolitis, immune and gut microbial dysregulation, and other complications. Diet and gut-microbiota-produced metabolites are sources of anti-inflammatories which could ameliorate symptoms. However, questions remain on how IBD influences biogeographic patterns of microbial location and function in the gut, how early life transitional gut communities are affected by IBD and diet interventions, and how disruption to biogeography alters disease mediation by diet components or microbial metabolites. Many studies on diet and IBD use a chemically induced ulcerative colitis model, despite the availability of an immune-modulated CD model. Interleukin-10-knockout (IL-10-KO) mice on a C57BL/6 background, beginning at age 4 or 7 weeks, were fed a control diet or one containing 10% (w/w) raw broccoli sprouts, which was high in the sprout-sourced anti-inflammatory sulforaphane. Diets began 7 days prior to, and for 2 weeks after inoculation with Helicobacter hepaticus, which triggers Crohn’s-like symptoms in these immune-impaired mice. The broccoli sprout diet increased sulforaphane in plasma; decreased weight stagnation, fecal blood, and diarrhea associated; and increased microbiota richness in the gut, especially in younger mice. Sprout diets resulted in some anatomically specific bacteria in younger mice, and reduced the prevalence and abundance of pathobiont bacteria which trigger inflammation in the IL-10-KO mouse, e.g., Escherichia coli and Helicobacter. Overall, the IL-10-KO mouse model is responsive to a raw broccoli sprout diet and represents an opportunity for more diet-host-microbiome research.

Importance

To our knowledge, IL-10-KO mice have not previously been used to investigate the interactions of host, microbiota, and broccoli, broccoli sprouts, or broccoli bioactives in resolving symptoms of CD. We showed that a diet containing 10% raw broccoli sprouts increased the plasma concentration of the anti-inflammatory compound sulforaphane, and protected mice to varying degrees against disease symptoms, including weight loss or stagnation, fecal blood, and diarrhea. Younger mice responded more strongly to the diet, further reducing symptoms, as well as increased gut bacterial richness, increased bacterial community similarity to each other, and more location-specific communities than older mice on the diet intervention. Crohn’s Disease disrupts the lives of patients, and requires people to alter dietary and lifestyle habits to manage symptoms. The current medical treatment is expensive with significant side effects, and a dietary intervention represents an affordable, accessible, and simple strategy to reduce the burden of symptoms.

Acknowledgements: This project was supported by the USDA National Institute of Food and Agriculture through the Maine Agricultural & Forest Experiment Station: Hatch Project Numbers ME022102 and ME022329 (Ishaq) and ME022303 (Li); the USDA-NIFA-AFRI Foundational Program [Li and Chen; USDA/NIFA 2018-67017-27520/2018-67017-36797]; and the National Institute of Health [Li and Ishaq; NIH/NIDDK 1R15DK133826-01] which supported Marissa Kinney, Timothy Hunt, and Benjamin Hunt. Johanna Holman was supported by ME0-22303 (Li), and Lola Holcomb was supported by US National Science Foundation One Health and the Environment (OG&E): Convergence of Social and Biological Sciences NRT program grant DGE-1922560, and the UMaine Graduate School of Biomedical Science and Engineering.

An American lobster on a countertop next to a ruler.

Lobster shell microbes, epizootic shell disease, and climate change manuscript is published!

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A collaborative paper on lobster shell bacteria has just been published in the journal iScience: “Water temperature and disease alters bacterial diversity and cultivability from American Lobster (Homarus americanus) shells.” This paper investigates what happens to bacterial communities on healthy and sick lobsters as they experience different water temperatures for a year.

You can read the paper here.

Woman sitting outside.

I joined this project back in the summer of 2020, towards the end of my first year at UMaine, when I was given a large 16S rRNA gene sequence dataset of bacterial communities from the shells of lobsters. I had been asking around for data as a training opportunity for Grace Lee, who at the time was an undergraduate at Bowdoin College participating in the abruptly cancelled summer Research Experience for Undergrads program at UMaine in summer 2020. Instead, Grace joined my lab as a remote research assistant and we worked through the data analysis over the summer and fall. Grace has since graduated with her Bachelor’s of Science in Neuroscience, obtained a Master’s of Science at Bowdoin, and is currently a researcher at Boston Children’s Hospital while she is applying to medical school.

My first point of contact on the project was Jean MacRae, an Associate Professor of Civil and Environmental Engineering at UMaine, who was the one to lend me the data and who had been working on bacterial community sequencing on other projects which I’ve been involved in. Jean has been involved with MSE, and this is our fourth publication together making her the collaborator at UMaine I have co-authored with the most (although it is a tight race 🙂 ).

Four professors wearing full regalia, as well as face masks, posing for a photo in a hallway.

Jean introduced me to the original research team, including Debbie Bouchard, who is the Director of the Aquaculture Research Institute and was researching epizootic shell disease in lobsters for her PhD dissertation several years ago; Heather Hamlin, Professor and Director of the School of Marine Sciences; Scarlett Tudor (not pictured), the Education and Outreach Coordinator at the ARI; and Sarah Turner (not pictured), Scientific Research Specialist at ARI. The ARI team is involved in a lot of large-scale aquaculture research, education, and outreach to the industry here in Maine, and the collaborative work I have been doing with them has been a new an engaging avenue of scientific study for me.

In 2022, the research team, along with social science Masters student Joelle Kilchenmann, published a perspective/hypothesis piece which explored unanswered questions about how the movement of microbes, lobsters, and climate could affect the spread of epizootic shell disease in lobsters off the coast of Maine. That perspective paper was a fun exercise in hypothesis generation and asking ‘what if’?

A steamed lobster on a plate.

This manuscript is more grounded, and features work that was started in 2016. It examines bacterial communities on the shells of lobsters which were captured off the coast of Southern Maine and maintained in aquarium tanks for over a year. The lobsters were split into three treatment groups: those which were kept in water temperatures that mimicked what they would experience in Southern Maine, colder water to simulated what they would experience in Northern Maine, and hotter water to simulate what they would experience in Southern New England over that year. The original project team wanted to know if temperatures would make a different to their health or microbial communities.

Figure S8. Water temperature regimes, related to STAR Methods. A. Temperatures were obtained through the National Oceanographic Data Center (NODC). NODC temperatures reflect those recorded near Eastport, ME (A); Portland, ME (B); and an average of temperatures from Woods Hole, MA (C) and New Haven, CT (D) was used to represent Southern New England. B. Annual temperature cycles used in this project to represent Southern New England (SNE), Southern Maine (SME) and Northern Maine (NME).

The original project team swabbed lobster shells to obtain bacteria to try and grow in the lab, as well as DNA to sequence and identify whole bacterial communities. Grace and I performed the data analysis to identify which taxa were present in those communities, what happened over time or when the water temperature changed, and what bacteria were present or not in lobsters which died during the study.

Figure S11. Lobster carapace sampling using a sterile cotton swab to obtain bacterial communities from the shell surface, related to STAR Methods. The right side of the dorsolateral area of the cephalothorax was sampled for the baseline sampling, the left side for the Time 1, and the right side again for Time 2.

In addition to wanting to know about temperature, we wanted to know specifically how temperature would affect the bacteria if the lobsters had epizootic shell disease. It is not known what causes epizootic shell disease (which is why it is called ‘epizootic’), but it manifests as pitting in the shells of lobsters. Over time, the pitting can weaken shells and make it difficult for the lobster to molt, or make the lobster susceptible to predators or microbial infections. This type of shell disease had been a huge problem in Southern New England over the past few decades, and in Maine we have seen more cases over time.

Four panels of lobsters showing the progression of a healthy lobster to ones with more and more pitting in their shells.
Figure S10. Examples of lobster shell disease indices, related to STAR Methods. A) 0, no observable signs of disease, B) 1+, shell disease signs on 1-10% of the shell surface, C) 2+, shell disease signs on 11-50% of the shell surface, D) 3+, shell disease signs on > 50% of the shell surface.

The highlights of this project are here, but you can click the link below to read the entire study and what happened to lobster health and lobster microbes over time.

  • Shell bacteria from healthy lobsters, often overlooked, were included in the study.
  • Hotter and colder water temperatures affected shell bacterial communities.
  • Epizootic shell disease reduced bacterial diversity on lobster shells.
  • Epizootic shell disease could be induced or exacerbated by the loss of commensal bacteria from shells.

“Water temperature and disease alters bacterial diversity and cultivability from American Lobster (Homarus americanus) shells.”

Suzanne L. Ishaq1,2,, Sarah M. Turner2,3, Grace Lee4,5,M. Scarlett Tudor2,3, Jean D. MacRae6, Heather Hamlin2,7, Deborah Bouchard2,3

  • 1 School of Food and Agriculture; University of Maine; Orono, Maine, 04469; USA.
  • 2 Aquaculture Research Institute; University of Maine; Orono, Maine, 04469; USA.
  • 3 Cooperative Extension; University of Maine; Orono, Maine, 04469; USA.
  • 4 Department of Neuroscience, Bowdoin College, Brunswick, ME 04011; USA.
  • 5 Boston Children’s Hospital, Boston, MA 02115; USA.
  • 6 Department of Civil and Environmental Engineering; University of Maine; Orono, Maine, 04469; USA.
  • 7 School of Marine Sciences; University of Maine; Orono, Maine, 04469; USA.

Summary

The American lobster, Homarus americanus, is an economically valuable and ecologically important crustacean along the North Atlantic coast of North America. Populations in southern locations have declined in recent decades due to increasing ocean temperatures and disease, and these circumstances are progressing northward. We monitored 57 adult female lobsters, healthy and shell-diseased, under three seasonal temperature cycles for a year, to track shell bacterial communities using culturing and 16S rRNA gene sequencing, progression of ESD using visual assessment, and antimicrobial activity of hemolymph. The richness of bacterial taxa present, evenness of abundance, and community similarity between lobsters was affected by water temperature at the time of sampling, water temperature over time based on seasonal temperature regimes, shell disease severity, and molt stage. Several bacteria were prevalent on healthy lobster shells but missing or less abundant on diseased shells, although some bacteria were found on all shells regardless of health status.

Catch of the day: lots and lots of microbiology

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Over the past few months, a large team of undergraduate, graduate, and postdoctoral researchers, and I, have been processing hundreds of samples from our scallop hatchery microbiome project. As 2022 winds down, so does the first phase of our lab work, and we are taking a well-deserved break over the holidays before we launch additional lab work, data analysis, and manuscript writing in 2023.

In 2021, the Ishaq Lab, collaborators at UMaine, and collaborators at the Downeast Institute ran a pilot project to investigate the bacteria that associate with sea scallop larvae in hatcheries, and how this is develops in relation to bacteria in hatchery tanks over time. For that project, we collected hundreds of culture plates with a specialized media that selects for certain species of bacteria.

When tanks are drained and cleaned every two days, cotton swabs are rolled across part of the bottom or side of the tank and used to inoculate bacteria onto these culture plates. This is part of a routine screening for pathogens, and don’t worry, we aren’t finding bacteria that causes disease in humans. But, these screening plates creates a useful starting point for our research on bacterial community dynamics.

Tank swab samples are used to inoculate TCBS plates to screen for Vibrio and similar bacteria

We received over 200 of these TCBS culture plates, and from them we isolated 140 bacteria in 2021 and early 2022 which we archived at -80 degrees Celsius. This was part of Sarah Hosler’s master’s of science thesis in August of 2022, and has since been passed to Ayodeji Olaniyi for part of his master’s of science thesis.

This fall, we were able to recover 115 of these isolates from the deep freeze, and tested them on 12 different media in duplicate, which created >1800 cultures plates and tubes, and 230 microscope slides!

This massive undertaking would not have been possible without a large team helping with the lab work, including rockstars Ayodeji Olaniyi, Sydney Shair, Keagan Rice, and Lacy Mayo who put in hours and hours leading the efforts on this. We are also grateful to Alaa Rabee, Aaron Williams, Lily Robbins, Ash VanNorwick, and Rebecca Kreeger who provided assistance with media making, inoculating, and the large amount of cleanup (we used glass or autoclavable plastic where possible, and sterilized some single-use plastics to be used as training tools for student education). We were also assisted by Bryanna Dube, who is working on creating outreach/education materials based on our results.

Now, our team will focus on analyzing the results of all these microbiology tests and look for trends. Some will also be heading to the Perry Lab to learn how to perform quantitative polymerase chain reactions (qPCR), in which we use a modified version of DNA replication to count the copies of specific genes. We will use this to look for genes which confirm the identity of our bacteria.

Beginning in summer 2022, the Ishaq Lab has also been part of a state-wide research and commercial collaboration to understand and improve sea scallop production in hatcheries and farms. As part of that project, we received 1500 DNA samples from different hatchery tanks or larvae over the summer/fall rearing season.

Gloria Adjapong is a Postdoctoral Fellow at the UMaine Cooperative Extension Veterinary Diagnostics Lab, and she has been graciously extracting these samples as part of her cross-training in the Ishaq Lab. We will use the extracted DNA to sequence the bacterial communities to identify which bacteria are present, and when, to understand microbial community dynamics over time and in relation to scallop health.

Alexis wraps up her REU project!

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Last week, undergraduate Alexis Kirkendall concluded her 10-week Research Experience for Undergraduates (REU) program at the University of Maine. Over that time, she has been an integral member of the Ishaq lab, and had assisted with lab work for multiple projects, comparing microscopy staining protocols, training students, assisting with laboratory management and safety regulation checks, and more. She picked up skills in animal sample collection, microscopy and staining, parasitology, culture media preparation, DNA extraction, and data visualization in R. Alexis also helped create some of the marketing materials for the Microbes and Social Equity Symposium in July, and facilitated group discussions as a note taker.

At the end of the program, REU students create posters and short presentations of their efforts over the summer. The presentations were last week, but you can check out the poster below.

Alexis is heading back to Ohio for her next year at Heidelberg College where, in addition to studying science, she is leading initiatives to make the campus more accessible and inclusive. But, we hope to see her back in Maine sometime in the future!

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Emily awarded an undergraduate research fellowship!

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The very first Ishaq Lab undergraduate researcher, Emily Pierce, has also been awarded the first fellowship of the Ishaq Lab!

Emily has been awarded a Faculty Fellows Research Assistantship for spring 2021 from the University of Maine Center for Undergraduate Research (CUGR)! The $1200 award will provide funds for salary to Emily and research materials, and will support her project for her AVS Capstone Experience (selected Capstone project summaries are here, but Emily’s is not included).

Portrait of Emily Pierce

Emily joined the lab in early 2020 to work on a project investigating calf health and gut microbes, but very soon after joining the lab, the SARS-CoV-2 pandemic emerged and changed the way we were able to interact on campus. Without missing a beat, Emily shifted her efforts from helping me wrangle the lab renovations and sorting out our inventory, to helping me improve my teaching materials, to diving deep into previous literature to dig up protocols for her experiment in 2021: “Ideal Conditions for Cryptosporidium Attachment and Infection.“

We’ll be performing the experiment itself over the winter break, and then using the spring to analyze the data and write them up. As part of the CUGR award, Emily will be presenting her work at the 2021 Student Symposium in April, which will be held virtually this year. You’ll have to wait till then to get more details!

(Reblog) A perspective on tackling contamination in microbial ecology

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Original posting from BioBE.

To study DNA or RNA, there are a number of “wet-lab” (laboratory) and “dry-lab” (analysis) steps which are required to access the genetic code from inside cells, polish it to a high-sheen such that the delicate technology we rely on can use it, and then make sense of it all.  Destructive enzymes must be removed, one strand of DNA must be turned into millions of strands so that collectively they create a measurable signal for sequencing, and contamination must be removed.  Yet, what constitutes contamination, and when or how to deal with it, remains an actively debated topic in science. Major contamination sources include human handlers, non-sterile laboratory materials, other samples during processing, and artificial generation due to technological quirks.

Contamination from human handlers

This one is easiest to understand; we constantly shed microorganisms and our own cells and these aerosolized cells may fall into samples during collection or processing.  This might be of minimal concern working with feces, where the sheer number of microbial cells in a single teaspoon swamp the number that you might have shed into it, or it may be of vital concern when investigating house dust which not only has comparatively few cells and little diversity, but is also expected to have a large amount of human-associated microorganisms present.  To combat this, researchers wear personal protective equipment (PPE) which protects you from your samples and your samples from you, and work in biosafety cabinets which use laminar air flow to prevent your microbial cloud from floating onto your workstation and samples.

Fun fact, many photos in laboratories are staged, including this one, of me as a grad student.  I’m just pretending to work.  Reflective surfaces, lighting, cramped spaces, busy scenes, and difficulty in positioning oneself makes “action shots” difficult.  That’s why many lab photos are staged, and often lack PPE.

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Photo Credit: Kristina Drobny

Contamination from laboratory materials

Microbiology or molecular biology laboratory materials are sterilized before and between uses, perhaps using chemicals (ex. 70% ethanol), an ultraviolet lamp, or autoclaving which combines heat and pressure to destroy, and which can be used to sterilize liquids, biological material, clothing, metal, some plastics, etc.  However, microorganisms can be tough – really tough, and can sometimes survive the harsh cleaning protocols we use.  Or, their DNA can survive, and get picked up by sequencing techniques that don’t discriminate between live and dead cellular DNA.

In addition to careful adherence to protocols, some of this biologically-sourced contamination can be handled in analysis.  A survey of human cell RNA sequence libraries found widespread contamination by bacterial RNA, which was attributed to environmental contamination.  The paper includes an interesting discussion on how to correct this bioinformatically, as well as a perspective on contamination.  Likewise, you can simply remove sequences belonging to certain taxa during quality control steps in sequence processing. There are a number of hardy bacteria that have been commonly found in laboratory reagents and are considered contaminants, the trouble is that many of these are also found in the environment, and in certain cases may be real community members.  Should one throw the Bradyrhizobium out with the laboratory water bath?

Chimeras

Like the mythical creatures these are named for, sequence chimeras are DNA (or cDNA) strands which are accidentally created when two other DNA strands merged.  Chimeric sequences can be made up of more than two DNA strand parents, but the probability of that is much lower.  Chimeras occur during PCR, which takes one strand of genetic code and makes thousands to millions of copies, and a process used in nearly all sequencing workflows at some point.  If there is an uneven voltage supplied to the machine, the amplification process can hiccup, producing partial DNA strands which can concatenate and produce a new strand, which might be confused for a new species.  These can be removed during analysis by comparing the first and second half of each of your sequences to a reference database of sequences.  If each half matches to a different “parent”, it is deemed chimeric and removed.

1024px-Splicing_by_Overlap_Extension_PCR.svg
Chimeric DNA

Cross – sample contamination

During DNA or RNA extraction, genetic code can be flicked from one sample to another during any number of wash or shaking steps, or if droplets are flicked from fast moving pipettes.  This can be mitigated by properly sealing all sample containers or plates, moving slowly and carefully controlling your technique, or using precision robots which have been programmed with exacting detail — down to the curvature of the tube used, the amount and viscosity of the liquid, and how fast you want to pipette to move, so that the computer can calculate the pressure needed to perform each task.  Sequencing machines are extremely expensive, and many labs are moving towards shared facilities or third-party service providers, both of which may use proprietary protocols.  This makes it more difficult to track possible contamination, as was the case in a recent study using RNA; the researchers found that much of the sample-sample contamination occurred at the facility or in shipping, and that this negatively affected their ability to properly analyze trends in the data.

Sample-sample contamination during sequencing

Controlling sample-sample contamination during sequencing, however, is much more difficult to control. Each sequencing technology was designed with a different research goal in mind, for example, some generate an immense amount of short reads to get high resolution on specific areas, while others aim to get the longest continuous piece of DNA sequenced as possible before the reaction fails or become unreliable.  they each come with their own quirks and potential for quality control failures.

Due to the high cost of sequencing, and the practicality that most microbiome studies don’t require more than 10,000 reads per sample, it is very common to pool samples during a run.  During wet-lab processing to prepare your biological samples into a “sequencing library”, a unique piece of artificial “DNA” called a barcode, tag, or index, is added to all the pieces of genetic code in a single sample (in reality, this is not DNA but a single strand of nucleotides without any of DNA’s bells and whistles).  Each of your samples gets a different barcode, and then all your samples can be mixed together in a “pool”.  After sequencing the pool, your computer program can sort the sequences back into their respective samples using those barcodes.

While this technique has made sequencing significantly cheaper, it adds other complications.  For example, Illumina MiSeq machines generate a certain number of sequence reads (about 200 million right now) which are divided up among the samples in that run (like a pie).   The samples are added to a sequencing plate or flow cell (for things like Illumina MiSeq).  The flow cells have multiple lanes where samples can be added; if you add a smaller number of samples to each lane, the machine will generate more sequences per sample, and if you add a larger number of samples, each one has fewer sequences at the end of the run. you have contamination.  One drawback to this is that positive controls always sequence really well, much better than your low-biomass biological samples, which can mean that your samples do not generate many sequences during a run or means that tag switching is encouraged from your high-biomass samples to your low-biomass samples.

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Illumina GAIIx for high-throughput sequencing.

Cross-contamination can happen on a flow cell when the sample pool wasn’t thoroughly cleaned of adapters or primers, and there are great explanations of this here and here.  To generate many copies of genetic code from a single strand, you mimic DNA replication in the lab by providing all the basic ingredients (process described here).   To do that, you need to add a primer (just like with painting) which can attach to your sample DNA at a specific site and act as scaffolding for your enzyme to attach to the sample DNA and start adding bases to form a complimentary strand.  Adapters are just primers with barcodes and the sequencing primer already attached.   Primers and adapters are small strands, roughly 10 to 50 nucleotides long, and are much shorter than your DNA of interest, which is generally 100 to 1000 nucleotides long.  There are a number of methods to remove them, but if they hang around and make it to the sequencing run, they can be incorporated incorrectly and make it seem like a sequence belongs to a different sample.

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DNA Purification

 

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Barcode swapping

This may sound easy to fix, but sequencing library preparation already goes through a lot of stringent cleaning procedures to remove everything but the DNA (or RNA) strands you want to work with.  It’s so stringent, that the problem of barcode swapping, also known as tag switching or index hopping, was not immediately apparent.  Even when it is noted, it typically affects a small number of the total sequences.  This may not be an issue, if you are working with rumen samples and are only interested in sequences which represent >1% of your total abundance.  But it can really be an issue in low biomass samples, such as air or dust, particularly in hospitals or clean rooms.  If you were trying to determine whether healthy adults were carrying but not infected by the pathogen C. difficile in their GI tract, you would be very interested in the presence of even one C. difficile sequence and would want to be extremely sure of which sample it came from.  Tag switching can be made worse by combining samples from very different sample types or genetic code targets on the same run.

There are a number of articles proposing methods of dealing with tag switching using double tags to reduce confusion or other primer design techniques, computational correction or variance stabilization of the sequence data, identification and removal of contaminant sequences, or utilizing synthetic mock controls.  Mock controls are microbial communities which have been created in the lab by mixed a few dozen microbial cultures together, and are used as a positive control to ensure your procedures are working.  because you are adding the cells to the sample yourself, you can control the relative concentrations of each species which can act as a standard to estimate the number of cells that might be in your biological samples.  Synthetic mock controls don’t use real organisms, they instead use synthetically created DNA to act as artificial “organisms”. If you find these in a biological sample, you know you have contamination.  One drawback to this is that positive controls always sequence really well, much better than your low-biomass biological samples, which can mean that your samples do not generate many sequences during a run or means that tag switching is encouraged from your high-biomass samples to your low-biomass samples.

Incorrect base calls

Cross-contamination during sequencing can also be a solely bioinformatic problem – since many of the barcodes are only a few nucleotides (10 or 12 being the most commonly used), if the computer misinterprets the bases it thinks was just added, it can interpret the barcode as being a different one and attribute that sequence to being from a different sample than it was.  This may not be a problem if there aren’t many incorrect sequences generated and it falls below the threshold of what is “important because it is abundant”, but again, it can be a problem if you are looking for the presence of perhaps just a few hundred cells.

Implications

When researching environments that have very low biomass, such as air, dust, and hospital or cleanroom surfaces, there are very few microbial cells to begin with.  Adding even a few dozen or several hundred cells can make a dramatic impactinto what that microbial community looks like, and can confound findings.

Collectively, contamination issues can lead to batch effects, where all the samples that were processed together have similar contamination.  This can be confused with an actual treatment effect if you aren’t careful in how you process your samples.  For example, if all your samples from timepoint 1 were extracted, amplified, and sequenced together, and all your samples from timepoint 2 were extracted, amplified, and sequenced together later, you might find that timepoint 1 and 2 have significantly different bacterial communities.  If this was because a large number of low-abundance species were responsible for that change, you wouldn’t really know if that was because the community had changed subtly or if it was because of the collective effect of low-level contamination.

Stay tuned for a piece on batch effects in sequencing!