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How to choose the size selection mode?

View: 92 / Time: 2022-02-18

Size Selection

Applicable Situation

Advantage

Disadvantage

Step 1

Optional steps:

Double-sided size selection after fragmentation

Sufficient DNA input;

Broad size distribution of DNA

fragments after fragmentation

Narrow size distribution,

high ligation efficiency and

uniform sequencing data

Loss of sample

Step 4

Optional steps:

Double-sided size selection after ligation

Sufficient DNA input;

Broad size distribution of

libraries after adapter ligation

Narrow size distribution,

Uniform sequencing data

Strict double-sized

selection parameters

based on library size


  • Loss caused by double-sided selection after fragmentation


Q6-1

Experimental conditions: 500 ng of Human Genomic DNA Male (Promega, catalog # G1471) was fragmented by using different enzymatic digestion times, and the procedures were conduct at 25℃ for 15, 20, 25 and 30min, respectively, then65℃ for 30 min. At the end of the fragmentation procedures, according to Appendix II 1.1 of the user manual, double-sided selection was used. After selection, Nuclease Free water was used to elute for subsequent adapter ligation.

This step of selection can make the fragmented samples more concentrated, thus leading to high efficiency of subsequent adapter ligation, but the sample loss is relatively high (about 60-70%), which is suitable for the library preparation of sufficient DNA. It is recommended that input DNA is ≥200 ng



  • Loss caused by double-sided selection after ligation


Q6-2

Experimental conditions: 100 ng of Human Genomic DNA Male (Promega, catalog # G1471) was fragmented by using different enzymatic digestion times. After the adapter ligation was

completed, the single screen mode and double screen mode were used respectively (see Appendix II 1.2 of the user manual for details).

The library yield after double-sided selection is 15-25% loss compared with  single selection. But it is not recommended for low-quality DNA or input DNA is ≤50 ng.

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