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 same controls are used, but at different concentrations.
The TruSeq Stranded RNA HT adapter plate is not compatible with TruSeq RNA Libary Prep Kits as the protocol and the reagents are different.
In the random priming process to generate cDNA with the best coverage, this information is not retained. If originating strand information is required, please refer to the Directional mRNA-Seq Sample Preparation Guide or other published applications.
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.
Illumina recommends that you check total RNA integrity following isolation using an Agilent Technologies 2100 Bioanalyzer with an RNA Integrity Number (RIN) value greater than or equal to eight.
Illumina uses a green laser to sequence G/T bases and a red laser to sequence A/C bases. At each cycle at least one of two nucleotides for each color channel need to be read to ensure proper registration. It is important to maintain color balance for each base of the index read being sequenced, otherwise index read sequencing could fail due to registration failure. For pooling strategies for a small number of samples, please refer to the Adapter Tube Pooling Guidelines section in the library prep guide for the kit you are using. Additionally it is recommended to create a sample sheet in the Illumina Experiment Manager (IEM) prior to performing sample prep in order to confirm appropriate index combinations not listed.
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.
The Illumina Experiment Manager (IEM) will notify you if improper combinations are used when creating a sample sheet for use with CASAVA, so it is highly recommended to create your sample sheet prior to performing sample prep/pooling. There are also low plexity pooling guidelines in the Nextera, TruSeq DNA, and TruSeq RNA sample preparation guides. Always pool samples with valid index combinations to avoid image registration failures.
The Directional mRNA-Seq Sample Preparation Guide provides instructions to use the Illumina Small RNA adapters, which are specific to the 3' and 5' fragment end, to generate a stranded library with the original Illumina mRNA-Seq kit. The protocol has not been optimized for the TruSeq RNA Sample Prep Kit and Illumina does not recommend this use of the TruSeq RNA Sample Prep Kit.
Longer drying times may be required depending on environmental variables and the amount of ethanol remaining in the well. However, take care not to over-dry beads as this can impact sample recovery.
TruSeq kits support many low plex pooling options across the entire plate. Some of these combinations are outlined in the Index Adapters Pooling Guide. If you designed your own color-balanced pools, Illumina highly recommends using the Illumina Experiment Manager to check the color balance of the pools.
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.
For kit compatibility information, see the Illumina Version Compatibility Reference.
Illumina does not support running libraries prepared by different library prep kits in the same lane of a flow cell. This practice may impact cluster density, data distribution, and run quality.
For runs on HiSeq Systems, creating and loading a sample sheet at the start of the run is optional. However, using a sample sheet allows you to view data shown on the indexing tab in the Sequencing Analysis Viewer (SAV) during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV.
If you are setting up a run in MiSeq Control Software or analyzing indexed samples using CASAVA v1.8.2, a sample sheet is required.
Illumina recommends that you create the sample sheet using the Illumina Experiment Manager (IEM) prior to performing library prep in order to confirm appropriate index combinations.
For TruSeq v2/LT kits, standard sequencing primers included in the cluster generation kits are required.
For TruSeq HT kits, primer usage will depend on the flow cell type used. Primers in the TruSeq Dual Index Sequencing primer kit, single read (catalog # FC-121-1003) are needed to run TruSeq HT Dual-Indexed libraries on single read v3 flow cells. These primers are HP10 (read 1), HP12 (Index read 1), HP9 (Index read 2 for single read flow cells only). This add-on box is not required if sequencing a TruSeq HT prepared library with the MiSeq System or on single-read rapid flow cells, or paired-end v3 or Rapid flow cells for HiSeq, HiScanSQ, GAIIx. The primers in the TruSeq Dual Index Sequencing Primer kits are backwards compatible with all Illumina libraries; there is no need to spike in primers as these primers are backwards compatible with other Illumina library types.
No. To enable demultiplexing, specify the correct use-bases-mask and use the appropriate sample sheet.
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.