I am having difficulty finding an answer to this question - what is the maximum DNA insert length feasibly permitted by MiSeq. By 'DNA insert', I mean EXCLUDING the adapters.
And does anyone know the the maximum DNA fragment length (ie INCLUDING adapters).
These terms seems to be used interchangeably, and when I see people referring to maximum lengths, I don't know which of the two they are referring to.
You don't find answer to this question is because there is no general consensus about the maximum length you can use. And the maximum DNA fragment length is always referred to "excluding the adapters + spacers + barcodes etc." However, they are considered while talking with respect to the Illumina sequencing strategy.
To decide the maximum length in your particular case, you have to consider about few things:
- What type of sequencing run you will be using, paired-end or single end sequencing?
- Do you want to pair/merge the reads if you have done the paired-end sequencing?
- What is you marker and how good it is to resolve the sequence diversity, or taxonomy in easier terms?
I have used a DNA fragment of 640 bp on MiSeq, which served our purpose very well. However, we have done paried-end sequencing (only terminology) or sequencing for both forward and reverse reads. Since the amplicon was too long to be merged, we had to use reads separately ; details can be found in this paper.Article High-Throughput Sequencing and Unsupervised Analysis of Form...
So, for you to decide what would be the DNA length in you case, you have to think over your marker. Our amplicon is sort of the border line because sequence longer than that would be difficult to sequence on MiSeq. Or you can think about any other platform link PacBio or Oxford nanopore in case its too long.
Illumina itself claims that a single read length can be up to 300 bp and use this value in some manufactured kits. However, I have seen many complains at researchers' forums around the world that there is a significant reading quality loss after about 200 bp, so the reads require trimming. I have also observed this in my practice. So, have in your mind: up to 150 bp - most probably good results; up to 200 bp - good chances of satisfactory results; up to 300 bp - depends on your skills, luck and research conditions (equipment, reagent batches and so on).
If you use a larger library size, you might have problems achieving optimal clustering during the sequencing. According to their library prep guide, https://www.illumina.com/documents/products/technotes/technote_nextera_library_validation.pdf, you can go up to 1000+ bp in library size. So that means an insert size of around 700bp, which about makes sense since mi-seq can do 300bp. The sequencing quality though, is another issue, for example see
https://www.nature.com/articles/s41598-019-39076-7
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