TruSeq DNA and RNA v1 kits are no longer available for order. Contact your local Account Manager for assistance in transitioning to the latest TruSeq RNA and DNA Sample Prep Kits for sequencing projects.
The TruSeq Stranded RNA HT adapter plate is not compatible with TruSeq RNA Libary Prep Kits as the protocol and the reagents are different.
TruSeq DNA and RNA Sample Prep kits - Set A
TruSeq DNA and RNA Sample Prep kits - Set B
While Illumina supplies additional indexed adapters in the TruSeq Sample Prep v2 kits, the fill volumes are kept at 8 samples to minimize disruption to the protocols. The v2 kits are limited by other components to 48 samples per kit. This also offers more flexibility for how customers use the indexed adapters.
For some library preparations, AMPure XP beads are user-supplied from Beckman-Coulter Genomics. See the appropriate TruSeq DNA or RNA library prep guide for more information.
Oligo-only kits are not offered for TruSeq DNA and RNA Library Preparation.
Resuspension Buffer should be stored at -25ºC to -15ºC when first received. After the initial thaw, the reagent can be stored at 2ºC to 8ºC for use throughout the protocol.
Illumina’s proprietary method ensures ligation of 2 different adapters in the required orientation to opposing ends of a DNA fragment. PCR selects for these and finalizes the construct ready for hybridizing onto the flow cell surface. Adapter sequences can be determined by sequencing the ligation fragments, but sequence information alone is not sufficient to uncover the method.
The number of PCR cycles should be minimized to avoid skewing the representation of the library. Illumina recommends 15 cycles of PCR, which has been shown to provide the best performance in terms of coverage, reproducibility, and quantity of material. Although not recommended by Illumina, if you want to eliminate PCR, begin with a large amount of starting material (~5 μg) to generate equivalent yield as the standard protocol. Note that at least one cycle of PCR is required to open the forked adapters.
Yes. To run the protocol without the controls, substitute Resuspension Buffer (RSB) for the control mixture at each step. See the appropriate TruSeq DNA or RNA sample preparation guide for details.
Yes. The Alternate Fragmentation Protocols section of the TruSeq RNA Sample Preparation v2 Guide (document # 15026495) describes the following options for varying the insert size of your library:
The DSN Normalization protocol was optimized for pre-TruSeq libraries. TruSeq RNA libraries are not readily amenable to the DSN treatment. There are two primary problems:
It is possible to use larger fragment sizes, however, we have found that shorter fragment sizes generate the best coverage. For more information, see Appendix A in the user guide for the TruSeq RNA library prep kit that you are using.
Samples should be quantified prior to pooling. It is possible to quantify after pooling if all DNA samples are of similar quality, but this requires very consistent yields and should not be attempted by a new user. See the appropriate library prep guide for details.
If pooling fewer than the number of indexes provided in the kit, it is necessary to consider low-plexity index combinations. Color balance during the index read is needed to ensure proper image registration. If there is no signal in one of the color channels (red or green) of the index read, image registration may fail and no base will be called for that cycle. If no base is called, the index read may not be able to be matched to the sequence specified in the sample sheet, and then samples will not be able to be demultiplexed. Refer to the Index Adapters Pooling Guide for recommendations and guidelines for Illumina sequencing systems that require balanced index combinations. Illumina Experiment Manager (IEM) gives warnings when generating sample sheets if the index combinations do not meet this requirement.
Yes, as long as the index read primer and multiplex read 2 primer are used. TruSeq DNA and RNA libraries have the same architecture and sequencing primer attachment sites as v2 multiplexed libraries.
The data is comparable to TruSeq RNA Library Prep v2 data. The TruSeq Stranded mRNA and Directional mRNA-Seq protocols are based on distinct ligation chemistries and the libraries they produce are expected to differ as a result. Direct sample-to-sample comparison is not recommended for these library types.
RTA 1.9 (on GA) or 1.10 (on HiSeq) or higher processes the controls. To visualize them, SAV (Sequencing Analysis Viewer) 1.7 or higher must be used. RTA produces a new InterOp metrics file called ControlMetrics; this is the file SAV uses to get control counts.