Sequencing information for Illumina DNA Prep (M) Tagmentation libraries

Which Illumina sequencing instruments are compatible with this library prep? These libraries can be run on all currently available Illumina sequencing systems. Should index reads be color balanced? Yes. Refer to the index adapters pooling guide to for appropriate index choice for the relevant sequencing platform.

Can libraries prepared in separate batches be pooled in the same run?* Yes, but all libraries must to be quantified prior to pooling.

• If input DNA was at least 100 ng, libraries from each independent experiment may be pooled and quantified together prior to pooling with the second batch.

• It is not recommended to pool all samples from independent experiments or batches without quantification.

Are data sets available? Yes, see BaseSpace Demo Data for public sequencing and analysis data (link requires BaseSpace login).

Which flow cell types can be used with these libraries? This library type is compatible with all Illumina flow cell types. Can Nextera XT index primers be used with Illumina DNA Prep library preparation? This is not recommended or supported. Illumina recommends using either the IDT for Illumina DNA/RNA Unique Dual (UD) indexes or the Nextera DNA CD index kits. Compatible index kits are listed below the Index Adapters heading on the product page. Why are the sequencing intensity profiles wobbly/spiky in the first ~15 cycles of the insert reads? This is a normal characteristic of tagmented libraries.

What is the recommended read length for this library type? 2x150 bp. Some instruments require shorter reads (2x149 bp) to account for the 10 bp dual indexes when IDT for Illumina DNA/RNA UD indexes are used. Can reads >2x151 bp be used on sequencers that have sequencing kits with >300 cycles (eg, the MiSeq, NovaSeq 6000, and NextSeq 1000/2000)? Longer reads lengths are not supported. Longer read lengths result in lower data quality with this library type due to the fragment size distribution of the libraries.

For any feedback or questions regarding this article (Illumina Knowledge Article #1263), contact Illumina Technical Support [e-mail].