Sequencing primer design for barcode sequencing

General rules and other notes:

1. Why do we need to add random N for the forward primer
5-7 cycles for R1 is used for both R1 and R2 to locate the cluster and color matrix during sequencing.
May need to use less dense clusters. Transitions from high to low diversity sometimes matter for getting a high sequence quality, as told by an Illumina scientist.

2. Spike-in guideline
The newer software allows us to forgo costly control lanes but instead use extra phiX.
For amplicons, at least 5-10% is needed to overcome low diversity issue

3. About random Ns/degenerate oligos/mixed base; (iDT Note)
When mixed bases are used, the oligo is a mixture of the bases in certain positions.

There are two ways of having random Ns, machine mix and hand mix.
machine mix: T is up to 30%
Hand mix: is about 24%
The length limitations for hand mix oligos is and was 100nt.
Machine mix will only do 25% each. Machine mix tends to over-represent G slightly and under-represent A.

Another technique is called "Ultramers" if we ever want to do long blocks.  Ultramers can go to 200nt, but all mixed bases are limited to machine mix(all equimolar).

There is such limitation for hand mix for 100 bp because,
The limitation is regarding iDT's standard synthesis; even with the highest coupling efficiency in the industry at about 99.5%, this means that the longer you go the lower the purity as you get more and more truncated species. At 98% coupling (like some of other DNA synthesis competitors of iDT) you are under 10% purity at 100 bases! For iDT, it gets to 0.995^100=60.6%.

Specifically, hand Mixed Bases provide customer-defined ratios of the desired bases, where coupling efficiencies are taken into account. For each mixture, IDT can incorporate up to 100 residues on the 100 nmole and 250 nmole scales and up to 40 residues on the 1 µmole scale, with a maximum of 4 unique Hand Mixes per oligonucleotide.

I personally think the cutoff doesn't make sense, especially when I don't care so much about purity but just want to have enough variants, but they just don't allow going over 100bp for synthesis. There are other ways to generate longer ones or stitch them together, but not as convenient.


Two designs of primers for having lower PhiX:

1. Staggering with different number of Ns for upstream primer
Illumina "if the amplicons will be of low diversity during read 2 as well, we will need to use a similar approach by staggering for read 2 as well or spiking in PhiX." -> Chuan: I don't think it is true, because I tried having just one end staggering and it worked fine, but I do believe having staggering at the read 2 would give better quality. Since we have extra length, let's just follow their instruction.

Illumina notes: These adapters look similar to our TruSeq dual-indexed libraries. I would expect these to cluster well on the flow cell and not require additional custom sequencing primers.
In the P5 end, you can remove the “universal index” sequence there, and that would look similar to our TruSeq single indexed libraries.

2. Multiple upstream and downstream primers with staggering lengths.
This design will ask for mixing multiple upstream and downstream primers but not having N. Careful calculation need to be performed to make sure we have a balanced base representation each time before we pick some primer.

The good thing is that we only need to do the calculation once. List it in an excel file and each time, we choose from the excel sheet.

We decide to proceed with design 1 because it sounds slight easier.

References and extra reading

1. Illumina technical note (2015)
https://support.illumina.com/content/dam/illumina-marketing/documents/products/technotes/technote-hiseq-low-diversity.pdf

2. Illumina notes for 16S metagenomic sequencing library preparation (similar to amplicon)
https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf
They used HiFi HotStart

3. Primer adaptors explained by Tufts University Core Facility
http://tucf-genomics.tufts.edu/documents/protocols/TUCF_Understanding_Illumina_TruSeq_Adapters.pdf

4. Illumina indexed sequencing
https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/miseq/indexed-sequencing-overview-guide-15057455-03.pdf

5. Illumina note about nucleotide diversity
https://support.illumina.com/bulletins/2016/07/what-is-nucleotide-diversity-and-why-is-it-important.html

6. Recommended PhiX percentages
https://support.illumina.com/bulletins/2017/02/how-much-phix-spike-in-is-recommended-when-sequencing-low-divers.html

7. a range of support bulletins from Illumina for future reference
https://support.illumina.com/bulletins.html

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