DNA quality control standards ratified for the Bennett Lab in Aug 2020:
Sequencing of whole plasmids or linear DNAs generally <25 kb. Requires no primers. Practically insensitive to extreme GC/AT content, structures, or repeats, except for homopolymeric repeats (single base stretch), which may be shortened by a one base.
Sequencing of 500–1000 ng plasmid or linear DNA in the region 800 bp after a primer that is provided or selected from the service's list.
Invented in 1977, Sanger sequencing is still the most common sequencing method (as of 2022).
Sanger sequencing reactions are generally reliable from 25 bp after the primer up to 800 bp, though good reactions can produce 1000 bp of good read. The sequence ≈25 bp downstream of the primer is generally of too poor quality to use, as is the sequence toward the end of the read. To cover a larger sequence, primers for multiple reactions must thus be designed in a way that either the ends of the sequencing reads are bound to overlap (convergent orientation of reads), and/or downstream primers overlap the reads of upstream primer's read (tandem orientation of reads). Quality scores are assigned to each nucleotide read of the chromatogram, so programs that read sequencing traces (.ab1 files) automatically grey out the low-quality regions and highlight mismatches and insertions/deletions. Ab1 files can be downloaded as part of sequencing results and uploaded for alignment to the template in Benchling.
See Lab Orientation page for instructions on Sanger sequencing.
Mechanism: A provided primer binds the sample DNA, is extended by a DNA polymerase in a reaction that contains a small amount fluorophore-labeled 2′,3′-dideoxynucleotide-triphosphates (ddNTPs) in addition to the normal dNTPs. Incorporation of a ddNTPs by the polymerase terminates the chain, as without a 3′ hydroxyl in the final dDNTP, the polymerase cannot extend the replicated DNA chain further. Because the ddNTPs incorporation is random (but still complimentary to the template DNA), a replicated chain of each and every length is produced by the polymerase extension reaction, and each length terminates in a complimentary ddNTP. Since each of the four ddNTPs have a unique fluorophore, fluorometric capillary electrophoresis of the reaction mixture will produce a sequence of fluorescence (a chromatogram) that corresponds to the sequence of the primer extension product from smallest to largest (closest termination from primer to farthest), which is the sequence of the template DNA.
For rapidly calculating volumes: in Wetlab Calculator, go to Sequencing by Mass, where at the top, enter desired DNA mass (e.g. 480 ng), reaction volume (6 µL), and primer volume (0.3 µL). For each sample, you need only enter the mass concentration as measured by Nanodrop, or the molar concentration if normalized. Then add the calculated water volumes to the sequencing tubes, then the common primer volume, and lastly the calculated DNA volumes (this lets you use one tip to quickly add a primer to all the clean water volumes).
$5.50 per sample.
Requested formula: 500, 800, or 1000 ng for <6 kb, 6–10 kb, and >10 kb plasmids. 10, 20, 40, or 60 ng for <0.5, 0.5–1, 1–2, and 2–4 kb PCR products. For larger PCR products, treat like plasmids. Combine with 25 pmol primer in a final 15 µL.
Simple formula: 2.5 µL 10 µM primer, ideally 500 ng DNA (as low as 300 ng), up to 15 µL with water.
In Wetlab Calculator, go to Genewiz Sequencing, where at the top, enter the desired DNA mass (500 ng), reaction volume (15 µL), and primer volume (2.5 µL). Then add the calculated water volumes to the sequencing tubes, then the common primer volume, then the calculated DNA volumes (this lets you use one tip to quickly add a primer to clean water volumes).
Benchling can run Primer3Plus for automated design of primers according to many parameters. Shyam spent a few hours fiddling with the parameters to get it produce primers like the ones he would design manually to optimally Sanger sequence the middle of long parts. These parameter calculations give primers with similar spacing and read overlap as meticulously manually-designed primers that split the target sequencing region into ≈equal portions covered by individual sequencing reads that overlap sufficiently not to leave any sequencing gaps. Primer3 has the added benefit of template specificity checking. It will generate pairs of divergent forward and reverse primers, but often you'll need only one of each pair to cover the entire target span with reads optimally spaced. –Shyam Bhakta
* Below, this 850 nt value represents your idea of reliable good sequencing read (after the ≈25 nt "junk" lead between the primer 3′ end and beginning of good sequencing trace). This spacing can be adjusted to the sequencing read length you're comfortable with, but be sure to adjust it in all the other parameters it's used here, marked with an * .
A paper that serves as a primer on outsourced NGS for the beginner: https://pubs.acs.org/doi/full/10.1021/acssynbio.1c00592
The focus on a protein variant library here can be generalized to any library.
Feb 2022 advice from Kshitij Rai (Caleb Bashor Lab).
Hack - My personal favorite hack is to leverage high scale read setups that commercial companies and cores have for different purposes (usually genome sequencing, or single cell RNA seq/bulk RNA seq). They don't understand targeted sequencing of a defined region with variability in the middle, as is the case with most syn bio projects. However, since companies run these services at scale, they give you really good prices (Genewiz charges ~$800 for 700 million reads). All you need to do is to get on the phone with these companies and tell them that you will be doing the library prep and that they just need to run it on their sequencer and give you the FastQ file outputs for your "genome" sample, and then prep and give them your sample. These services are very, very cheap, and open up a lot more options. Baylor's sequencing core is one I would really recommend for this purpose. I believe they offer 200 million reads for $350 or something ridiculous like that, and will give you results in ~1 week.
Low - Medium Depth (2-10 million Reads) | ||||
Sr. No. | Provider/Company | Read Depth | Cost | Comments |
1 | Genewiz | 15 million | $1344 | Quote requested |
2 | Wyzer Biosciences | 4 million | $1120 | Single end reads |
3 | Beijing Genomics | NA | NA | Do not offer a service at this depth |
4 | MD Anderson Sequencing Core | 15 million | $1708 | |
5 | DNA Link Sequencing Lab | 50 million | $249 | Paired end reads, 100bp max |
6 | LGC Biosciences | 50 million | $1826 | |
7 | ABM | NA | NA | Did not respond to calls or emails |
High Depth (200-400 million Reads) | ||||
Sr. No. | Provider/Company | Read Depth | Cost | Comments |
1 | Genewiz | 350 million | $1440 | Additional 30% off on first NGS Run |
2 | Beijing Genomics | 300 million | $700 | Best price |
3 | LGC Biosciences | 200 million | $5843 | Quote requested |
4 | MD Anderson Sequencing Core | > 100 million | $3238 | Costs only $1632 for MD Anderson faculty |
5 | DNA Link | Quote requested | ||
6 | ABM | NA | NA | Did not respond to calls or emails |