qPCR quantification using our new Agilent Bravo robot

We have an exciting new instrument in the Genomics Core which will enable us to automate several of our protocols which until now have been quite labour intensive. This is the Bravo Robot by Agilent. After some previous experience with automation, I think that the results we have seen up until now are really quite promising with the aim of saving hands on time and providing consistency and accuracy within protocols.

qPCR test run - One of the first tests we ran upon installation of the Bravo was qPCR quantification. We quantify all libraries which are submitted to our sequencing service so we can aim to generate cluster densities on the flowcell which will yield large amounts of high quality data.

For this test, a single RNA-seq library was quantified using our standard method with the Illumina quantification kit by Kappa Biosystems. To test the reproducibility of the robot, we performed qPCR on this one sample 24 times as this is the maximum number of tubes which can be loaded per run.

In addition to this, each of the same 24 aliquots of this one library was set up in the same way but manually. This test was useful to determine how good the liquid handling on the Bravo is by looking at the reproducibility of the 24 replicates but additionally for comparing that between manual versus automated set up. The library had also been quantified previously so we expected the concentration to be 50nM.

Agilent Bravo Robot

The results show that there is higher variation in concentrations achieved manually although both methods slightly overestimated the expected concentration of 50nM. We saw an average concentration of 55.6nM manually (an 11% increase from 50nM) in comparison to a concentration of 52.7nM (a 5.5% increase from 50nM) on the Bravo.

Despite there being an about 1.8% difference between the average concentrations seen on the manual set-up in comparison to that of the automated, we can see that the Bravo has yielded more consistant results which is what we would expect and also good news. Since this test, we are now quantifying all SLX library submissions using the Bravo.

Although the robot will not be for general use and we will be unable to run individuals qPCR, we will be using it for all qPCR quantification for libraries submitted to us for our NGS service and for generating RNA-seq libraries. Once these protocols become robust within our lab, we will explore the option of utilising the robot in other protocols including Exome libary prep. It is additionally going to be used for automation of ChIP by the Odom group.

Help! I really don’t know anything about Next Generation Sequencing…

Over the last three years, while managing the CRUK-CI NGS service, I have heard one phrase more times than I can count:

Help! I really don’t know anything about Next Generation Sequencing…

Yet I have not got bored of hearing this phrase, or of explaining the basics of NGS to fellow researchers and colleagues. That is quite simply because (i) I think NGS is the bee’s knees, and (ii) my answer is never the same twice.

I understand why NGS can be daunting to researchers who wish to use it for the first time; it is technically complex at the same time as being unforgiving if you make a mistake – quite frankly, it is difficult to do well, and expensive if you screw up. The principles underlying the Illumina SBS chemistry are very simple, but the vagaries of instrumentation, the concepts behind designing a good experiment, and the technical skill required to create a high quality library add a layer of complexity which can frighten entire labs away from NGS.

The one thing I always open with is “Don’t be put off; it’s not as complicated as it looks”. The rest of the story changes every month, as new technologies are released and old ones are updated or improved.

Regardless of the name of the current Illumina big-hitter, or the details I advise you take into account when using a particular kit, there are some tips that I give to everyone, which I can also share with you. I will be honest and say they are unashamedly Illumina-focused. I manage an NGS service which runs Illumina sequencers, what else did you honestly expect?

1. Start with the Basics

I have not yet found a better resource for NGS training than the Illumina website (let's overlook the rubbish search function and confusing navigation for the time being). The user-training videos are easy to follow but sufficiently comprehensive to get you started. Furthermore, you can re-watch them once you have some experience, to help with troubleshooting. Don’t even start your experimental design until you have understood how the process works, or you will make expensive mistakes.

Illumina’s training site: http://support.illumina.com/training/sequencing_training.ilmn

Start with

Sequencing: Illumina Technology

MiSeq: Sequencing Fundamentals

TruSeq: DNA PCR-Free Sample Prep or TruSeq: Stranded RNA Sample Prep

Truseq: Best Practices

Later on, consider

Sequencing: Illumina’s Dual Indexing Strategy

MiSeq: Data Analysis Overview

MiSeq: Imaging and Base Calling

Truseq: Sample Purification Bead Size Selection and Best Practices

Linux Introduction

2. Practice, Practice, Practice

This should be obvious, but you might be surprised how many folks get it wrong. Don’t ever do your first NGS library preparation with your precious, tender, super-rare, pride-and-joy samples! Don’t even think about it! Don’t expect to buy one 96-sample kit and get 96 high quality NGS libraries with your first prep. Start small. Practice until it works, and then perform your experiment.

When you are ready to try NGS for the first time: buy a small kit, and source a batch of DNA or RNA that you can waste while you learn how to make good libraries. Start by making 8 libraries for practice, following the kit instructions very carefully. Where possible, collect a small quantity of DNA/cDNA after each cleanup step which you could use to troubleshoot failures.

I would strongly recommend that you sequence your best batch of practice libraries, rather than relying solely on the library QC. It is easy to make an RNAseq library which looks quite convincing on the bioanalyzer but performs poorly in differential gene expression analysis, and very, very easy to make a library which looks like a nice small RNA library which contains only the Illumina adapter sequence.

3. It’s Not What You Know, it’s Who You Know

It is unlikely that your first adventure in NGS will be plain sailing, so it’s important to know where you can go for help. If you’re new to NGS and you’re based here at the CRUK Cambridge Institute, the best starting point is to come down to the Genomics Core and talk with us. We’re always happy to meet NGS novices and to help you get your experiments started. If you’re not based in the CRUK CI I’m afraid we can’t offer you much help – we’re a small team and we’re dedicated to supporting this Institute. If you can find a Genomics Facility in your institute then they are going to be your best bet for introductory information, as well as more targeted advice.

If you’re not lucky enough to have a dedicated team onsite then the next best resource for you is Illumina’s technical support team (techsupport@illumina.com). They are well-informed and always happy to help with troubleshooting.

Other great sites to help you troubleshoot your NGS problems are:

http://seqanswers.com/

http://www.nature.com/nrg/series/nextgeneration/index.html

http://www.nature.com/nbt/focus/sequencing/index.html