Increasing Your Digital Profile

A central tenet of the Technician Commitment is Visibility; to ensure that all technicians within the University of Edinburgh are identifiable and that the contribution of technicians is visible within and beyond our University. The University has an important role to play here ensuring that technicians are represented on websites and publicity as well as supporting policies to include more technicians on publications and grants. In this blog I will focus on how we can take responsibility for our own digital footprint to increase our visibility to colleagues and across our institutes.

1) Get an ORCID iD (https://orcid.org/)

An ORCID iD provides every researcher with a unique identifier. It allows easy identification of researchers and their publications, which is particularly important if you have a common name. It is a free service and is easy to set up. Once you have an ORCID iD you can use the information to then populate other research tools (such as Pure, see point 2).

Technicians should be on papers and by having an ORCID iD you are showing you are expecting to be included on papers that you are contributing to. It also makes it easier to be added on to papers and your research administrator will then add you as using your ORCID iD profile will mean your pure profile will automatically update with your newest publications.

A year on, what’s changed?

2021 here we are. How has your working life changed over the last year?

Here at the Genetics Core, like so many other workplaces across the UK, we closed our doors in March 2020. A surge of planning began; how do we work from home? What is expected of us? How do we adapt our laboratory jobs? Although contingency planning had begun well before we went into lockdown, I don’t think it really prepared us for what lay ahead. Our meetings had gone from all of us onsite to people dotted around living rooms, kitchens, hallways and staircases. The dreaded mute/unmute fiascos had started, children curious to see what mum or dad were doing, pets greeting the postie, the list goes on and on.

As we settled down into our work from home lives we set our sights on reviewing, updated and writing our Standard Operating Procedures (SOPs), Risk Assessments (RAs) and Control of Substances Hazardous to Health (COSHH) assessments – a task that was always pushed aside as we would run off into the lab with purpose!

Using our time to focus on the Core development; researching automation for both our sample processing team and sequencing team, website development, continuing the development of the Genetics Core’s improved training competencies. The ‘Tech Session’ was created and technicians were asked to present protocols to the team in order to reaffirm their learning while educating others. This time allowed technicians to work together to make informative and interactive learning experiences for the whole team, creating a document that would allow trainees to look to for a clear understanding of a process.

As we all worked away, the front line staff were hard at work helping to save lives. Our Research Nurses moved to front line patient care, while Professor Kenny Bailie, an intensive care Doctor, expanded his already existing GenOMICC project (Genetics of Mortality in Critical Care). The GenOMICC project was created to allow research of future pandemics and had all of the ethics and SOPs in place to rapidly allow a greater understanding of critical illness in COVID patients. At this point, in March 2020, we had 100 samples from patients. As the project opened up to COVID sufferers in critical care, the numbers of research samples grew rapidly, with Intensive Care Units (ICU) across the UK signing up to help contribute to the ongoing medical research effort. For more information about the GenOMICC project, please visit their website here; GenOMICC.

Breaking Out The CAGE Or, continued adventures in Cap Analysis of Gene Expression

The lockdown put in place as a result of the Sars-CoV-2 pandemic has brought about many changes in the way people live and work. The Genetics Core closed only briefly before reopening to allow the processing of samples as part of a UK wide initiative to sequence the whole genomes of patients to identify the specific genes that cause a predisposition to the disease. Information on this project led by the multi-talented Dr Kenny Baillie can be found here.

It was members of Dr Baillie’s research group (Ivet Gazova and Sara Clohisey) who came to the Genetics Core to provide training in CAGE library preparation, having used the technique most recently in an investigation of transcriptional activity during Influenza A virus infection (Clohisey et al 2020). As we’re all now spending at least some time working from home – to limit the numbers in the lab and ensure social distancing – it seemed fitting I take a moment to write an update on work I described in a previous blog.

To briefly recap, I adapted an established protocol for Cap Analysis of Gene Expression library preparation that would allow sequencing on our Illumina NextSeq 550. This was on behalf of Dr Emily Clark, a Chancellor’s Fellow at the University of Edinburgh Roslin Institute leading the BBSRC-funded Sheep Gene Expression Atlas Project, a key contribution to the international Functional Annotation of Animal Genomes (FAANG) Initiative. The development work was supported by Dr Jeffery Schoenbeck, also a Chancellor’s Fellow at the Roslin Institute specialising in canine genetics and genomics, morphology and disease and heritable traits.

A pilot run with new barcoded linker oligos and sequencing primers worked on the NextSeq but discussions with Mazdak Salavati, the bioinformatician analysing the data, revealed the protocol needed further optimisation. I set about redesigning the linkers with an extended barcode sequence to allow a higher level of multiplexing and improved demultiplexing of each pooled library from the sequence data.

Acknowledging the Genetics Core

The College of Medicine & Veterinary Medicine (CMVM), University of Edinburgh, has a policy for acknowledging the important contribution core facilities make to research within CMVM. This was circulated by email, but I have not seen a copy online, so I have attached it below for reference.

What does it say?

It outlines what is expected from researchers based in CMVM, when acknowledging core facilities that they have used. This covers all publications such as papers, posters and presentations. For those researchers within CMVM this is policy and so is compulsory; for those researchers outside the college it is considered best practice.

Why is it important?

Core facilities are important (I would say that, I run one). The Covid19 situation has shown just how important, with core facilities often the last area of institutes to close and the first to re-open. As research budgets come under strain it will be more important than ever that we make maximum utilisation of expensive equipment. That we provide equitable access and we support the technical experts that can get the most out of these platforms.

Most core facilities within CMVM operate by a cost-recovery model. This can lead to the mistaken belief that the relationship with a core facility is transactional and that the usual scientific norms of acknowledgement or authorship in a publication is not required. This policy document makes clear that this is not the case. Core facilities need these publication metrics as much as any researcher, to prove their worth and ensure they remain viable. Including core facilities during the writing phase can also improve your publication, especially the method section.

What do I need to do?

Read the policy below and think about how you will acknowledge the core facilities you have used. If that includes the Genetics Core and you have any questions please do get in touch (Lee. Murphy @ ed.ac.uk). And when you have acknowledged the Genetics Core please do send on your paper, we love hearing about your work and how we have supported you. This feedback really helps with our training, making us better scientists and better at supporting you.

Due to the cost-recovery model and the extended Covid19 closure many core facilities will be facing holes in their budget and your support is needed now more than ever.

Let’s all do our best to support our fantastic core facilities.

 

 

The NextSeq 2000 is here! First impressions.

The sequencing team at the Genetics Core are lucky to have landed the brand new sequencer from Illumina, the NextSeq2000. As of this moment (early May 2020) due to the Covid-19 situation, we are one of only a few labs in the world to have it up and running with real samples! Of course, this is great news, however, it means we’ll probably come across some of the typical glitches that come with brand new machines and software.

The new NextSeq2000

Our IIlumina FAS, Clare Logan, kindly took the time to do a presentation from the comfort of her home (courtesy of the lockdown). She then visited the lab to assist us with our first sequencing run using libraries made from RNA extracted from whole blood from Covid19 infected patients.

For this first sequencing run we produced a good amount of data for a P2 flow cell. The run generated 122Gbp with a >Q30 of 89% and 570M reads passing filter. Not bad for a first run as Illumina’s specs are 400M reads passing filter and 120 Gbp output when using a P2 flow cell.

Digital PCR using the Naica™ Crystal Digital PCR™ System from Stilla

Digital PCR (dPCR) is an end-point PCR method that offers an alternative to conventional real-time qPCR for absolute quantification of nucleic acids and rare allele detection. The method is similar to qPCR in terms of the reaction components (primers and labelled probe detect sequence specific targets) and the subsequent amplification, but differs in the way the sample target is measured.

In dPCR the nucleic acid sample mix is partitioned into many individual PCR reactions (droplets or wells, see Figure 1). Then amplification of the individual reactions is conducted and each well or droplet will be either positive (1) or negative (0) for the target molecule.  The number of positive vs negative reactions is determined and permits the generation of an absolute count of the number of target molecules in the samples.

This simple and reproducible method, does not rely on a calibration curve for sample target quantification, therefore no reference standards or endogenous controls are needed allowing for greater robustness and reproducibility among different labs. Poisson correction is applied to account for individual reactions that may have more than one molecule of target sequence present. The history of dPCR is discussed in the following article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129430/ and the technology is reviewed here: https://www.ncbi.nlm.nih.gov/pubmed/29677144.

RNA sequencing options

What is RNA-seq?

RNA Sequencing (RNA-Seq) uses massively parallel next generation sequencing to detect and quantify the RNA from a sample.

Whilst there are a variety of RNA-Seq applications and protocols, most follow the basic strategy of isolating RNA converting it to DNA and then adding adapter sequences to generate a library suitable for sequencing.

The first step in any RNA-Seq project begins with identifying the experimental question, i.e.  What are you interested in studying? To answer that, we need to look at what RNA-Seq can be used for.

 

What are the applications of RNA-seq?

Whole Transcriptome Sequencing

Whole transcriptome sequencing enables the characterization of all RNA transcripts including both the coding mRNA and non-coding RNA regardless of polyadenylation. To accurately look at the whole transcriptome, most library preparation protocols first start with the depletion of ribosomal RNA (rRNA) which otherwise takes up the majority of all sequencing reads.  Assuming you’re not interested in ribosomal RNA, removing these transcripts allows for more of the sequencing reads to be focused on transcripts you’re actually interested in sequencing, giving you improved sensitivity toward low expressed transcripts.

mRNA Sequencing

mRNA-Seq is an RNA-Seq protocol that enriches for polyadenylated (PolyA) transcripts of the transcriptome. mRNA-Seq is a method used to study transcription in disease states as well as expression in variety of research based applications. Only around 1-2 % of the entire transcriptome is comprised of PolyA tailed RNA which is the coding part of the genome. By targeting mRNA, sequencing depth is improved as resources are dedicated to the sequencing of coding genes. This makes identifying rare variants and low expressed mRNA transcripts easier.

Gene Expression Profiling

Gene expression profiling measures which genes are being expressed in a cell at any given moment. Gene expression profiling identifies differentially expressed transcripts among groups of samples. Typical analysis includes mapping reads against a reference, counting reads per transcript, and statistical testing for differential expression.

Small RNA Sequencing

Small RNA Sequencing is a technique to specifically isolate and sequence small RNA species, such as microRNAs (miRNAs). Mature miRNAs are naturally occurring, 22-nucleotide, non-coding RNAs that mediate posttranscriptional gene regulation. Alterations in miRNA can be correlated with gene expression changes in development, differentiation, signal transduction, infection, aging and disease.  Unwanted RNA species will be removed by size selection during library prep to ensure that a high proportion of reads represent the small RNAs.

Single Cell RNA-Seq

Single cell RNA-Seq (scRNA-Seq) is a tool that enables simple and robust access to the transcriptomes of thousands of single cells. This method enables analysis of millions of single cells in a high-throughput manner.

Top 5 tips for looking after your freezers

Freezers need to work hard to keep important samples and reagents at a safe temperature. Ultra Low Temperature (ULT) freezers are also one of the most energy hungry items in the laboratory, using up to £1,000 of energy each year. By looking after our freezers and following these five tips we can ensure sample integrity and have a positive impact on the environment.

1) Give them space

To work efficiently a freezer needs to have a constant flow of air. Generally cool air is pulled through filters at the front and bottom of the freezer to the back where heat transfer occurs at the condensers, the warm air then rises and re-enters the room. To help the air flow freezers should not be pushed up too close to the wall, or to each other, so try to leave at least a 30cm gap at the back and sides. Most importantly, nothing should be stored on top of the freezer, a mistake that happens in many labs.

Air conditioning can help heat exchange and limit how hard the freezer compressor is working but this comes at an environmental cost. Before turning on the air conditioning consider if a lower tech solution is available i.e. open a window!

Equality, Diversity and Inclusion: A Technician Lens Report Launch

I was given the opportunity to attend the STEMM Change Equality, Diversity and Inclusion: A Technician Lens report  launch at the Royal Society of Chemistry on the 14^th of November 2019, as a representative of the University of Edinburgh Technician Steering Committee (TSC) . This event was a standalone component of the STEMM Change annual conference, which was a wonderful event looking at Equality, Diversity, and Inclusion within academia, however as a last-minute substitution to attend to represent the TSC, with travel to London only being booked on the 11^th of November (EEK!), I only made it to the afternoon sessions.

Climate Strike (#ClimateStrike)

On Friday 20th September, my eldest son and I joined in a global day of action. Fridays for Future (#FridaysForFuture), has become a massive movement orchestrated by young people around the world, who feel that world leaders are still not taking sufficient action to mitigate the effects of man-made climate change.

I wholeheartedly agree, and as a scientist, it is frustrating that despite the extremely vast pool of scientific data people are still questioning climate change.

I particularly enjoyed this paper by Cook et al., entitled ‘Consensus on Consensus’ https://iopscience.iop.org/article/10.1088/1748-9326/11/4/048002/pdf which goes into this issue in depth and concludes that the 97% consensus that Global climate change is caused by human activities and that it is occurring now and that it is a threat to society has been confirmed and reconfirmed and reconfirmed!

I think that means it’s time to stop questioning and start acting. As a mother of 2 young children, I am deeply afraid that if we don’t take strong action now, my boys are going to pay for it. So, if there is anything that I can do now to try and stop this happening – I’m certainly going to do it.