Expiration dates are printed on the kit box labels and reagent tubes. Illumina guarantees three months of shelf life from the time of shipment.
A sample sheet is required prior to starting a sequencing run and enables the correct identification and processing of libraries after the sequencing run is streamed to BaseSpace Sequence Hub. Use IEM v1.8 or later and select the TruSeq Synthetic Long-Read DNA kit and the HiSeq FASTQ Only application.
The TruSeq Synthetic Long-Read DNA Library Prep and Barcode kits are designed for two applications; preparing DNA libraries for de novo synthetic long-read sequencing and analysis and phasing analysis from whole human genome sequencing data. The kits combine TruSeq and Nextera chemistries to prepare DNA libraries for sequencing and informatics apps to aid in performing long reads assembly or phasing analysis.
The TruSeq Synthetic Long-Read application generates at least 600 MB of synthetic long reads (>1.5 kb). The number of barcode plates prepared depends on the target genome size and the depth of the existing build. A reasonable starting point might be 5–10x genome coverage of synthetic long reads for genome finishing.
Illumina recommends 50 μl input DNA at 10 ng/μl.
For more information, see DNA Input Recommendations in the TruSeq Synthetic Long-Read DNA Library Prep Guide.
Use a fluorometric-based method for quantification, such as Qubit or PicoGreen, to accurately quantify dsDNA.
UV spectrophotometric-based methods, such as the NanoDrop, measure any nucleotides present in the sample, including RNA, dsDNA, ssDNA, and free nucleotides. This measurement can give an inaccurate measurement of gDNA.
The expected range for final, pooled libraries is 200–3000 bp when assessed on a Agilent Technologies 2100 Bioanalyzer using a High Sensitivity DNA chip. Illumina has observed a wide range of concentrations (20–200 nM) depending input gDNA quality, operator technique, and other variables.
Quantify final libraries using a fluorometric quantification method that uses dsDNA binding dyes or qPCR.
Qubit measurements are substantially higher because it measures total DNA while qPCR measures ligated, amplifiable fragments. To make sure that the appropriate amount of DNA is used for long-range PCR, accurately quantify the DNA using qPCR, then use the value to calculate dilutions.
No, they can be stored at 2°C to 8°C for up to 3 months. Freezing long-fragmented DNA can lead to significant strand breakage leading to poor results, such as low or no yield, and short N50.
A full list of user supplied items can be found in the Consumables and Equipment section of the TruSeq Synthetic Long-Read DNA Library Prep Guide or TruSeq Synthetic Long-Read DNA Library Prep consumables and equipment excerpt. Comparable performance is not guaranteed when using alternate equipment.
Yes, a sample sheet is used to specify how each lane is to be analyzed. However, it is critical that TruSeq Synthetic Long-Read DNA library sequencing parameters (2 × 101 + 8 bp index) apply to the entire flow cell. If multiple TruSeq Synthetic Long-Read DNA libraries are run on the same flow cell, each must be run on a separate lane because the same barcoding scheme is utilized for each sample.
A minimum of 30 GB of short-read data is recommended. Data sets < 30 GB result in a warning. Analysis of < 30 GB data might result in poor phasing or assembly results.
The maximum input is 115 GB of short read data. Exceeding this limit results in a warning.
Purchase of the kit includes access to TruSeq Long-Read Assembly or TruSeq Phasing Analysis applications in BaseSpace.