Illumina DNA Prep library preparation for the MiSeq i100 Series 25M Reagent Kit (1000 cycles)

Illumina has released the MiSeq i100 Series 25M Reagent Kit (1000 cycles) to enable 2x501 bp sequencing on the MiSeq i100.

Illumina has performed testing to increase the library sizing for Illumina DNA Prep libraries prepared from high quality genomic DNA to support this longer read configuration. The following provides information for this application of Illumina technology that has been demonstrated internally and may be of interest to users. This information is provided as‐is, is not an official Illumina protocol, and is not accompanied by any rights or warranties. Users of this information should obtain any licenses required and materials from authorized vendors. Illumina products mentioned herein are for research use only. While feedback is welcomed, this application is not supported by Illumina Technical Support and Field Application Scientists.

The Illumina DNA Prep Reference Guide, which includes the full supported protocol is available on the page here.

To support the 2x501 bp read configuration, the following protocol modifications have been tested by Illumina development.

Input

Use a minimum input of 100 ng. Lower input mass may lead to low library preparation yield and is not recommended.

Protocol changes

For the Amplify Tagmented DNA section of the protocol:

The standard protocol recommends 5 cycles of PCR for input between 100 ng and 500 ng. In the context of this modified protocol, use 7 cycles of PCR for input between 100 ng and 500 ng.

For the Clean Up Libraries section of the protocol:

Use a modified double-sided bead clean up. Modifications are indicated relative to the standard protocol steps by number, with the changes bolded. Any steps not included below are performed per the standard protocol.

1. Transfer 47.5 µl supernatant from each well of the PCR plate to the corresponding well of a new MIDI plate.

...

1. For standard DNA input > 500 bp, perform the following steps.

a. Add 42.5 µl nuclease-free water to each well-containing supernatant.

b. Add 38.7 µl IPB to each well-containing supernatant.

...

g. Transfer 126.2 µl supernatant from each well of the first plate into the corresponding well of the new MIDI plate containing 3.75 µl undiluted IPB.

Additional protocol steps

Libraries must be manually normalized prior to pooling. Illumina recommends pooling at least 24 samples per pool to have sufficient library mass for subsequent steps. After pooling the normalized libraries, perform a size selection of the pool with Blue Pippin (Sage Science, product BDF1510) using 30 µl of the pool.

For the Blue Pippin size selection, follow the protocol and consumable recommendations from the manufacturer, here. Size select for the 1000-1500 bp range of fragment sizes.

Check library pool

Final yield following the Blue Pippin selection is expected to be 1-2 nM, with average library size between 1000 bp and 2000 bp.

Example TapeStation trace of a final library pool, run with a High Sensitivity D5000 ScreenTape Assay.

Sequencing

Illumina has used a final loading concentration of 120 pM, though the appropriate loading concentration must be empirically determined by each lab.

In sequencing these longer libraries, Illumina has observed sequencing output of 20-25 million reads per run, with ≥85% Q30. Sequencing runs with libraries prepared with this modified protocol are not guaranteed to meet Illumina performance specifications.

In sequencing, a G overcall has been observed due to the presence of some shorter library fragments. Illumina has observed this increase in G call starting approximately 120 cycles into each insert read and reaching an approximate 10% overcall. With trimming, the G overcall is not expected to impact data usability. An example of the G overcall pattern in the percent base plot is below.

The A overcall spikes in the last cycle of each insert read are not informative and should be bioinformatically trimmed prior to downstream data analysis. To trim these last cycles, use Override Cycles Y500N1;I10;I10;Y500N1 in the sample sheet/planned run with BCL Convert.

For any feedback or questions regarding this article (Illumina Knowledge Article #10102), contact Illumina Technical Support [email protected].