Best/Cheapest Services for DNA Sequencing & Synthesis

[Patrik D'haeseleer]

Hi all,

So where does an independent DIYbio lab find the best deals when it comes to DNA sequencing and synthesis? Market places like Science Exchange offer tons of services that we could avail ourselves from, but figuring out which is the best deal is often less than trivial. So let's put together some of our own favorites here...

We may want to skip the small stuff - ordering single primers, or getting some Sanger sequencing on a PCR product has become so commoditized that you can just about find them on any street corner. So let's look at some of the more cutting-edge applications:

• Who offers the cheapest / most convenient bacterial genome sequencing?

• Who offers the most affordable RNAseq services?

• Who offers the cheapest / most convenient 16S, ITS or other DNA barcoding services?
  

• What is the cheapest human genome sequencing available to DIYbio?
  

• Who has the best rate for buying relatively large numbers of 60nt oligos (e.g. for DNA origami)?
  

• What is the best rate we can find for longer oligos (>100nt, e.g. to encode text messages)?

• What is the best rate for gene synthesis, and longer DNA?
  

Please post your favorites!

Patrik

[Dakota Hamill]

https://www.wyzerbio.com/wiser/#aboutUs  has been extraordinarily kind to my friend and I.  

--
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diy...@googlegroups.com. To unsubscribe from this group, send email to diybio+un...@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To unsubscribe from this group and stop receiving emails from it, send an email to diybio+un...@googlegroups.com.
To post to this group, send email to diy...@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/7b6f629e-f871-4d08-b77c-73faa08fdac3%40googlegroups.com.
For more options, visit https://groups.google.com/d/optout.

[Patrik D'haeseleer]

I'll post some of my own favorites to start:

- uBiome (of sequence-your-poop fame) offers microbiome sequencing for $89/sample, no preprocessing required. Of course, they're aimed primarily at human microbiome, but you can send them pretty much any mixed bacterial sample, and they'll do 16S sequencing and send you the results. Not sure if they can do ITS for mixed fungal samples as well; worth talking to them about that.

- IDT's gBlocks is currently one of the best deals to get smallish genes synthesized: $89 for 500bp, up to $329 for 2000bp!

[Patrik D'haeseleer]

On Friday, January 9, 2015 at 4:38:44 PM UTC-8, Dakota wrote:

https://www.wyzerbio.com/wiser/#aboutUs  has been extraordinarily kind to my friend and I.  

Sounds like they mostly do Sanger though - any bacterial genome sequencing?

Which services would you recommend them for?

[Dakota Hamill]

I think they offer more than their website shows, but don't quote me on it.  They're two guys who left a larger company to start their own thing.  Maybe Avery can chip in his 2 cents.  They gave my friend and I a bunch of free sequencing because at the time we couldn't afford it, they offered to help hone PCR parameters, did PCR cleanup, primer optimization, and sequencing editing.  They also had same day, or next day if they were really busy, turnaround time, even for two kids who sent in 5-10 samples at a time.  

They just seemed to bend over backwards to help us whether we shipped them 1 sample, or 1000.

I don't  know what other services they offer as we only used them for sequencing, but they blew GeneWiz out of the water.

--

-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diy...@googlegroups.com. To unsubscribe from this group, send email to diybio+un...@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To unsubscribe from this group and stop receiving emails from it, send an email to diybio+un...@googlegroups.com.
To post to this group, send email to diy...@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio.

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/f4f50d9c-7954-4dd6-8750-a3ceac45eb53%40googlegroups.com.

[Koeng]

I can second IDT gBlocks for being best for small genes

In my experience, genescript is very very good at complex genes. If you want to synthesize like a terminator or a CRISPR repeat, they can do it. I once got a 30bp inverted repeat separated by about 20bp, which was notoriously hard to sequence, but they still got it out of production faster than I could actually succeed in sequencing it after I got it. Their service is quick, and I've used them a couple times without any hassle. Recommended!

I believe gen9 has cheaper synthesis rate... but I've had a lot of problems with them in the past

IDT has the best rates for over 60bp IF you buy in bulk... For smaller orders invitrogen does much cheaper ~100bp primers. But beware! They are cheaper but not as pure. (In my experience, havent tried many other companies)

For text messages, I think gBlocks are best for price

-Koeng

[Patrik D'haeseleer]

Any recommendations for bacterial genomes?

You should be able to multiplex 96 genomes on a single MiSeq lane, so the cost will be mostly determined by library generation. rather than the sequencing itself. 

It'd be great if there was some service where you can order just a single bacterial genome sequenced, library generation and all. Most NGS sequencing facilities aren't going to want to put up with the overhead of selling 1/96th of a lane though...

[Nathan McCorkle]

On Jan 9, 2015 10:28 PM, "Patrik D'haeseleer" <pat...@gmail.com> wrote:
> It'd be great if there was some service where you can order just a single bacterial genome sequenced, library generation and all. Most NGS sequencing facilities aren't going to want to put up with the overhead of selling 1/96th of a lane though...

It wouldn't be a bad thing to get 96X though, or at least get that price to do order pooling. Ideally the pooled order would still get library generation treated on their end too. But the right person could pick that up as part of the pooling and as source of income if they could do the prep reliably.

[Jeswin]

Can you explain what you mean by a single MiSeq lane? Our lab has Life
Tech Ion Protons so I'm a little familiar with that. It's not my area
so I only know a little bit about the NGS setup. I know my colleagues
do exome seq on the chips, and I think they can run 16 (or was it 8 or
4, can't remember) different samples on a chip using barcode adapters,
but I think that limits how many reads they can do per sample. As long
as the customer is willing to work with us, I think we try to tackle
most projects, being a startup company and all.

[Patrik D'haeseleer]

On Saturday, January 10, 2015 at 8:53:45 AM UTC-8, phillyj wrote:

Can you explain what you mean by a single MiSeq lane? Our lab has Life
Tech Ion Protons so I'm a little familiar with that. It's not my area
so I only know a little bit about the NGS setup. 

Sorry, I meant HiSeq lane of course, not MiSeq (the MiSeq flowcell is essentially a single lane). But even a Miseq should be able to multiplex four bacterial genomes or so on a single cell.

The HiSeq does two flowcells per run, with 8 lanes per cell. Mick Watson has a good analysis here:

The rapid-run mode of the HiSeq 2500 is perfectly capable of producing 150 million 150bp paired-end reads. This equates to 45Gb of sequence data.

If we are sequencing 5Mb genomes, at 40X, we need 200Mb of sequence. 96 of those will therefore need ~20Gb of sequence, so as you see, a single lane of HiSeq 2500 easily copes.

ARK-Genomics runs a non-profit full cost recovery business model, which means we charge for reagents, staff time and equipment. So for that lane of sequencing, we would probably charge in the region of £2500.

We need to factor in the cost of libraries. In reality, we could make this cheaper via automation, but for the sake of ease, let’s say the library prep is £100 per sample. That’s £9600 on library prep.

That’s a total of £12100, or £126 per genome.

In reality, I think we could get library prep down to £50 per sample, This would bring the cost down to £76 per genome.

Given that that was written in 2013 and I'd expect costs have gone down slightly since, you can probably translate £ to $ and not be all too far off. But even at £126 per genome, that'd be only $190 for a de novo sequenced bacterial genome, library prep included. That's an awesome deal, provided we can find a supplier that is offering that kind of service to us DIYbio folks!

Patrik

 

[Nathan McCorkle]

On Sat, Jan 10, 2015 at 10:42 PM, Patrik D'haeseleer <pat...@gmail.com> wrote:
>That's an awesome deal,
> provided we can find a supplier that is offering that kind of service to us
> DIYbio folks!

Seems like it is potentially an e-mail (to ARK) and kickstarter
campaign away from being a reality. Maybe some special
sample-collection mailer kits? Pushing all the prep to the trusted
lab, along with being able to mail the sample directly to the lab
would seem to encourage under-funded academics to participate as well,
people who might not have a lot of sample to waste.

[Patrik D'haeseleer]

Speak of the devil - I just got an email promotion through ScienceExchange for de novo genome sequencing and assembly for $1500 per sample, at a sequencing depth of 100x for smaller genomes (up to 5Mb).

That's definitely more expensive than it needs to be, considering how many of these you could multiplex on a single Illumina run, but as far as I know that's still the lowest (or *only*?) published rate for bacterial genome sequencing. And entirely feasible for a DIYbio lab with a little crowdfunding. For that price, they do handle all the multiplexing and demultiplexing logistics, and provide genome assembly as well.

Here's the email I got, complete with discount code at the end:

De novo Assembly of Novel Genomes: $1,500 per Sample At 100x Coverage
Next generation sequencing technologies provide high genome coverages at a relatively low cost. However, due to limited read lengths, de novo assembly often leaves gaps or fails to distinguish circular genomes from linear. myGenomics can help you fill this gap by combining standard library prep (PE100) with a single (3-5Kb) mate-pair library followed by sequencing at high depth coverage all for a special promotional price of $1,500 per sample. The mean sequencing depth coverage for this service is >100x for smaller genomes (up to 5Mb). Sequencing is performed on an Illumina platform and data analysis of an assembled genome (FASTa file) is included.

Experienced Team of Ph.D Scientists
myGenomics is a CLIA lab that specializes in next generation sequencing and is based in Alpharetta, Georgia in the United States. All work is done on site to ensure the highest quality and the fastest turnaround possible.    

Easy to Submit Samples and Get Results
Please send a sample of 1-5 μg high-quality human DNA, blood or saliva samples packed with blue ice packs. The turn-around time is 20-30 business days. Data analysis if needed is available for $200/sample and will deliver VCF (variant call file), CNV and cnLOH reports with BWA mapping and GATK variant call information. Included filters are variant allele frequency (1000 genome), known pathogenic variants (dbSNP) and suspect variants (frameshift, stop gain, stop loss), SIFT score, Polyphen score.  You will also receive various deliverables such as sample QC, fragmentation QC, library prep QC, raw data (FASTq files), sequencing reads QC, coverage and FastQC plots.

Redeem This Offer
To start, simply request a quote and include the promotion code "NovelGenome-mG" in the Promotion Code field of your request. A volume discount is available for >10 samples. The offer ends at the end of September, but you just need to request a quote to qualify. You can work with the lab members on the project after this date.  

Here's the company on Science Exchange (listed as $1650/sample without the promotion code):

https://www.scienceexchange.com/labs/mygenomics-llc

If anyone knows of a better deal, please post them here!

Patrik

[Koeng]

"Idea: Community effort genome seq?

Andreas, our friend on the list, recently asked about using the Oxford Technologies Nanopore. I reached out because the lab I work for does have a Nanopore MiniIon. He proposed sequencing thePanellus stipticus genome to determine how to synthesize the fungal luciferase. I don't know if the sequence is publicly available or not. I like his proposal but I will have to convince the boss to carry this out. I don't really know how similar the Nanopore is to the Ion Proton NGS platform, in terms of cDNA library preparation or data analysis. It will be wonderful if the community came together to sequence an organism. I don't know how it can be done. If we can sequence the genome and make it public, I think it will be a big step for DIYbio. Time is of the essence for me."

This is pretty relevant^ and could probably be easily done.

-Koeng

...

[Nathan McCorkle]

On Fri, Aug 14, 2015 at 12:15 PM, Koeng <koen...@gmail.com> wrote:
> "Idea: Community effort genome seq?
>
> Andreas, our friend on the list, recently asked about using the Oxford
> Technologies Nanopore. I reached out because the lab I work for does have a
> Nanopore MiniIon. He proposed sequencing thePanellus stipticus genome to
> determine how to synthesize the fungal luciferase. I don't know if the
> sequence is publicly available or not.

Seems they only know the mechanism now, and that a 35kDa hydroxylase
is involved... maybe more has been found since this was announced:
https://groups.google.com/forum/#!topic/diybio/WVqcdjwgcRc
(which mentions, among others, "The Chemical Basis of Fungal
Bioluminescence", Angewandte Chemie International Edition, Volume 54,
Issue 28, pages 8124–8128, July 6, 2015)

[Patrik D'haeseleer]

Looks like Panellus stipticus is already sequenced though:

http://genome.jgi.doe.gov/Panst_KUC8834_1_1/Panst_KUC8834_1_1.info.html

Patrik

[Josiah Zayner]

I think it really depends on what you are actually after.

Do you want just a sequenced genome(i.e. raw data)? A sequenced and assembled genome? Are you going to do library prep and DNA extractions? &c.

Here is a good primer for most of what you need to know about bacterial genome sequencing: https://peerj.com/preprints/453.pdf

For sequencing there are a number of places in the bay area that will pick up sequences from you and charge a few $ per sequence. At NASA we use Sequetech and some people at Biocurious use Elim Bio.

For DNA "barcoding" generally just using Sanger sequencing is the way to go. Most places give discounts if you are doing lots of samples.

For RNAseq, again it depends on how much work you want to do and what you want to do and who you trust. From what I hear Oregon State Core has really good prices for external users at ~$1500/ lane but I am sure local universities (Stanford) might also do something similar?

If you plan to do 16s (V3 or V4) microbiome sequencing I think Argonne National Lab in Illinois has the best prices in my not comprehensive but general search. That is if you plan to do analysis yourself and you have them do everything else(DNA extraction, library prep, sequencing). Here is a quote from an email chain that I had with Dr. Sean Gibbons, he has worked on lots of the popular press microbiome sequencing projects with Dr. Jack Gilbert at UChicago and Argonne:
-----

If you want to get quantitative, I suggest a minimum of 3 replicates, and a maximum of ~8 reps (if you have the money to burn - going higher than that probably won't gain you much). I think paying for library prep is generally a good idea, both in terms of cost and data quality. The people working at the facility should be good at what they do, and produce consistent libraries. If the facility screws up, then they'll have to redo it. If you screw up, then you have to eat the cost. Also, buying your own barcoded primers is expensive (depending on how many primers you need this can be hundreds to thousands of dollars), not to mention your time. All in all, I think paying for the prep is the best bargain.

Here's a cost breakdown for Illumina MiSeq sequencing facility at Argonne:

DNA extraction: $14.25 per sample
PCR/library prep (16s): $6.63 per sample
1 MiSeq Sequencing Run (paired-end, 151X151 base pairs; can pool ~300 samples in one run): $2243.72

Therefore, if we assume 300 samples:

DNA Extraction: $4,275.00 (this is a step you could easily do yourself to save $$)
PCR and library prep: $1,989.00
Sequencing: $2,243.72

The above will give you 10,000-100,000 sequences per sample. I believe the MiSeqs can yield ~250bp in a single direction now. I tend to not use the reverse reads, since they are usually lower-quality, and you don't gain a whole lot more phylogenetic info with assembled forward-reverse reads. I tend to use the V4 region of the 16S gene, and the first 150 base pairs are the most phylogenetically informative.

You can probably go cheaper by sequencing on an Illumina HiSeq. Also, I'm not sure what the prices are like at other facilities. I like Argonne because I get consistent results.

As far as advise is concerned, you may want to sequence a few negative controls (run the DNA extraction on sterile water, and send this sample to the facility). Whatever shows up in the negative controls can be filtered from the rest of your data as contamination (either contaminants in your reagents, or from processing at the sequencing facility).
-----

Thanks,
     Josiah Zayner