The PacBio Sequel is up and running at RTLGenomics. Installed in October of 2016, this instrument provides much more data (4-5x) as a SMRT cell on the RSII for just around 1/2 the price for the equivalent amount of data. This means you can complete your project for significantly less money and time. With upcoming chemistry and software releases we expect the Sequel to exceed the capability of the RSII. Contact us for more information about sequencing on the PacBio Sequel or for generating a hybrid genome using PacBio and Bionano data.
Are sequels better than the original? We sure think so!
One year after announcing our new PacBio RSII, RTLGenomics is excited to announce the installation of the PacBio Sequel™ System. As the next platform in SMRT sequencing technology, the Sequel System offers the long read capabilities expected from PacBio technologies with a few added perks. The Sequel System has one million zero-mode waveguides (ZMWs) compared to the RSII at 150,000 ZMWs. This dramatic increase offers higher throughput and potential decrease in sequencing time. Coupled with this, the Sequel generates 7x as many reads as the RSII. The ability to generate high quality, long reads of high consensus make the Sequel System ideal for clients interested in whole genome sequencing, RNA-Seq, or finishing a previously sequenced genome.
Similar to the RSII, input material for the Sequel System needs to be high quality intact DNA. A comfortable concentration range for submitting DNA is 5ug-10ug. We also recommend assessing genomic DNA integrity using a Tapestation or Fragment analyzer. At the minimum, we recommend running an agarose gel.
As most of you know, we have been unable to run amplicons longer than about 400bp since early 2015. The cause of this was an issue with the Illumina reagents which resulted in a steep drop off in quality of the last 100bp for read 2 on a 2x300 kit. Once we discovered the source of the problem we were forced to move all of the orders already received to a shorter amplicon and restrict new orders to shorter amplicons as well. This of course was not an ideal situation as it reduced the number of variable regions available to sequence, and made it difficult (if not impossible) to compare between orders run on a long amplicon previously.
As you can imagine the whole situation was very frustrating for us as we know it was for many of you. Illumina was pretty accommodating and tried their best to mitigate the negative effects. However, we still had delays and slowdowns because of the situation. The good news is, it looks like we are going to be able to start offering the long chemistry again. In late March we had a conversation with Illumina and they informed us they believed the issue was resolved. We have run two test runs with the updated chemistry and our initial reviews show that the Q-score’s stayed at a consistent level throughout the run with only the normal gradual decline instead of the steep drop off we’d seen all last year. The reads are stitching together well, and we are very optimistic moving forward.
Moving forward we will be accepting orders for longer amplicons (up to 530bp) and have already started running the orders that had been on hold for the longer chemistry. If you’ve done work with us prior to 2015 you know our favorite amplicon is 28F-519R – and now we get to use it again! There will be a period of transition as we move forward. Many of our current orders still require the shorter runs, and to maintain consistency with previous data we will run 2x250 for them. Our plan for the near future is to run one 2x300 run per week as we start transitioning back to longer assays.
If you have questions about the new amplicon options, or just want to know how this will affect you, please don’t hesitate to contact us.
For many people the only good mouse is a dead mouse; however for RTL researcher Dr. John Hanson that is definitely not the case. In a new article in Zoological Studies (PDF) Dr. Hanson and an international team of collaborators demonstrated the existence of an undescribed species of fish-eating rat, and named it after an expert in the group.
The new species, Neusticomys vossi, is part of a very specialized group of South and Central American rodents called the Ichthyomyini (fish rats). Despite their name most of the species in this group are small (mouse sized), rodents that live in and around fast moving streams – usually near small waterfalls and rapids. They are very rare in collections due to this unique lifestyle. Generally, when field biologists are trying to catch rats and mice, they use traps baited with grain and/or fruit or peanut butter. These tend to be irresistible to most rodents. However, the Ichthyomyini are generally uninterested in this form of bait. This is because they tend to be more carnivorous or insectivorous. According co-author Dr. Thomas Lee of Abilene Christian University – the researcher who caught the new species – the best way to catch these mice is to “find a stream and put the trap in the middle of the water”. It seems the rodents are more attracted to the insects that wash into the trap or come in to it for the regular bait than they are to the actual bait.
The interesting thing about this new species is its existence was first suggested by the scientist it was named after, Dr. Robert Voss, Curator of Mammals at the American Museum of Natural History. In a 1988 monograph Dr. Voss noted a difference in size of mice from the eastern slopes of the Andes in Ecuador – but didn’t feel there was enough evidence to separate them. However, after Dr. Lee caught the fresh samples, Dr. Hanson was able to use molecular techniques to sequence part of the genomes of both eastern and western Andes mice. The genetic evidence clearly separated them into two groups.
With the rapid rate of change we find in all areas of the world’s biosphere, but especially in areas of South America’s highlands, documenting and cataloging biodiversity is critical. Knowing that Neusticomys vossi is a different species from similar mice on the western slopes of the Andes provides land managers in Ecuador and Colombia more data for planning, but also provides a data point that future scientists can look back to as they try to describe the change in biodiversity over time.
Ever wanted to do long-read sequencing of genomic material? Maybe you’ve needed a closed microbial genome or full transcript length sequences to identify isoforms. Well never fear – now you can do all that and more. At the beginning of November Research and Testing Laboratory brought in-house PacBio sequencing online. Although RTL has offered PacBio Sequencing for over a year, up until now we were forced to subcontract that service to another lab. This led to significant delays and higher costs. However, now that we have the machine in house we expect to be able to provide high quality sequences in a fraction of the time.
If you aren’t familiar with PacBio that’s ok – it isn’t a very common sequencing technology. However, its utility is starting to become more apparent and the number of researchers who use it is increasing. The benefits of PacBio are it allows for long reads (10kb or more) that easily read through stretches of the genome that other technologies struggle to get through (long repeats, high CG regions). This data allows researchers to close contigs that wouldn’t normally be easy to close. In addition since most PacBio libraries are created without amplification they still have the methylation information included on the DNA – which can be recovered without expensive, labor intensive steps. Of course this is mainly cost effective for smaller genomes (bacteria and some fungi) as the coverage required for higher eukaryotes is somewhat restrictive.
If you are planning on doing a PacBio run here are a few things to keep in mind. First, the best input material is high quality intact DNA. It doesn’t make a lot of sense to use highly fragmented DNA if you are trying to get long reads. Second, it takes a lot of DNA to make a library. Libraries can be made with as little as 1ug but you will have lower success. Standard amounts are 5ug-10ug of DNA. Third, samples are sequenced on SMRT cells. Each SMRT cell generates about 50k reads, with an average read length of 10kb. There will be smaller fragments, but there will also be much longer fragments (80kb is possible). Fourth, for smaller bacterial genomes you can generally close the genome with a single SMRT cell. For eukaryotic genomes you will need 20-200 SMRT cells to close a genome. Many people will generate hybrid genomes, using PacBio data to build a 5-10x scaffold and underlying that with 50-80x Ilumina data.
It is also possible to do amplicon sequencing on the PacBio using fusion primers. This is great if you are looking at a long amplicon (1kb-10kb) and want to get it in a single read – however because the through put of the PacBio is lower than other machines it is more expensive per read.
One last thing – in the past PacBio has had a bad reputation for generating low quality reads. Although this is still the case if you look at individual reads– the consensus reads that are able to be bioinformatically processed can in many cases have much higher Q-scores than any other chemistry. So if you are interested in long reads but are worried about the quality of PacBio (or if you just want more information on setting up an order) feel free to contact us for more information and a more technical explanation. As always the best way to get a hold of us is through firstname.lastname@example.org
Let’s get to sequencing!
Research and Testing Laboratories was founded in 2004 to aid researchers like yourself in exploring the human microbiome and the role of biofilms in human infection. We were one of the initial players in NexGen sequencing and the development of amplicon pyrosequencing techniques starting in 2008 when we acquired our first Roche 454 sequencer. In the eight years that followed we have expanded our NexGen sequencing offerings to include numerous Roche 454, IonTorrent PGM, IonTorrent Proton, Illumina MiSeq, Illumina HiSeq, Oxford Nanopore Minion and PacBio RSII sequencers. As our sequencing capacity and expertise has grown, we have developed into a well-known and trusted genomics company working with researchers in over 50 countries around the world. In recognition of this we have begun the process of moving all of our genomics and metagenomics services under a new division, RTLGenomics. Over the coming months be prepared to see changes to our website, documentation and email addresses as we make this transition. Rest assured that we are still the same great company with the same great people that you have worked with. Any services offered by Research and Testing Laboratory in the past will still be available through RTLGenomics. We here at RTLGenomics would like to thank you for all your business throughout the years and we hope to continue working hard for you in the years to come!