Is it possible to select any insert for Illumina sequencing?

The choice of the right insert size is an important part in planning your sequencing experiment. Size selection is typically done after fragmentation of the input DNA and adapter ligation, using gel electrophoresis or beads. The choice of possible insert sizes is limited by technical factors of the sequencing process, in Illumina SBS especially by the bridge amplification step. To read more about the different parts of a prepared DNA fragment please take a look on the figure in the article [link to “Why do get reads of the same length when you sequence differently sized fragments (Illumina)”].

What is the optimal insert size?

For Illumina systems DNA insert size range of 200–800 bp. It depends on the used Illumina system, the used reagents (kits) and the mode (single/paired end). That's why you should first take a look in the operating instructions of your used device and kit. For this reason there is no general recommendation and we can only give you some examples shown in table 1.

Illumina System Supported insert size
NextSeq 550 bp (with NextSeq v2 reagents)
350 bp (with NextSeq v1 reagents)
HiSeq X 350 bp (with HiSeq X Reagent Kit v2) 450 bp (for TruSeq Nano DNA and TruSeq DNA PCR-Free libraries)

Figure 3: Examples for supported insert sizes in different Illumina Systems

Is there a maximum fragment sequence length (DNA insert) for Illumina?
Yes. As we know by now, and the insert size limitation becomes an issue when you want paired-end reads with a higher inner distance with fragments longer than 800 bp. As an alternative, there is a preparation method called „Mate pair sequencing“ which enables you to use fragments up to 5 kba. Or you could look into modern techniques of long-range sequencing such as 10x Genomics and others.


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Last updated on May 18, 2019

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