Libraries prepared with the following sample preparation kits at the GGBC can be used on any of Illumina’s next generation sequencing instruments including MiSeq, NextSeq, and NovaSeq. Note that a substantial variance in reads per library is to be expected when pooling genomic libraries obtained from diverse samples with varied genome sizes.
Crucial recommendations for all samples
- Use fluorometric based methods for quantification (Qubit or PicoGreen) of the template DNA instead of UV spec based methods (e.g., NanoDrop) to obtain accurate DNA measurement.
- DNA should have absorbance ratio values of 1.8–2.0.
- RNA samples can be suspended in RNase-free water or 1X TE buffer prepared with RNase-free water.
- RNA integrity should be assessed using the Agilent Bioanalyzer (or any similar system).
- Please add any QC information (Qubit data, Agilent run, fragment analyzer run, gel picture,…) available to the online order form.
- If you are are submitting 8 or more samples, the samples must be in a 96-well plate and include an Excel file with the sample layout in your order.
- PLEASE USE V BOTTOM PLATES AND NOT ROUND/FLAT BOTTOM PLATES.
RNA is a sensitive material. RNA should be kept on dry or wet ice during transportation to the GGBC. Wet ice is recommended only for samples that are being transported locally within Athens. If you are traveling from farther away to submit your RNA samples, keep them on dry ice during transit. The GGBC will not accept RNA samples that are not stored properly during transit.
NGS stranded or unstranded RNA library preparation (Illumina compatible)
In the stranded libraries the vast majority of the sequencing reads are from the first strand. Selecting certain library types depends on the application. Generally speaking, all expression-by-sequencing analyses should use stranded libraries. Non-stranded RNA libraries can also be prepared upon request. Feel free to contact us at email@example.com to discuss your project.
Currently at GGBC, we use the Kapa Biosystems RNA library preparation chemistry for constructing of stranded libraries. We prepare both single- and dual-indexed libraries, depending on the target level of multiplexing. The quality of starting RNA is crucial for making good libraries and most importantly for robust data analysis. Starting material can be:
- 100 ng – 4 μg of total RNA in < 50 µl
- 10 – 400 ng of rRNA depleted or poly(A) – enriched RNA
RNA samples can be suspended in RNase-free water or 1X TE buffer prepared with RNase-free water. RNA integrity should be assessed using the Agilent Bioanalyzer (or any similar system). For sample’s concentration, we prefer using fluorometric methods, such as Qubit or Ribogreen.
NGS ribo-depleted stranded or unstranded RNA library preparation (bacteria)
Treatment of the total RNA from bacteria with the Ribo-Zero protocol removes cytoplasmic (nuclear-encoded) rRNA (5S, 16S, and 23S) prior to strand-specific or unstranded library preparation and sequencing. Please provide 1-5 µg of total RNA in RNase-free water.
TruSeq small RNA library preparation
The TruSeq Small RNA sample preparation kit primarily targets microRNAs and other small RNAs, that have a 5’-phosphate and a 3’-hydroxyl group, to generate cDNA from total RNA or purified small RNA. Up to 48 samples can be multiplexed in one sequencing lane. Please provide 1 to 20 μg of high-quality total RNA prepared using a method that retains the small RNA fraction (Trizol etc.) at a concentration of at least 250 ng/µl in high quality water or 10 mM Tris buffer. Alternatively, submit the entire fraction of small RNA purified from 5-20 μg of total RNA in molecular grade water or 10 mM Tris buffer. Starting with enriched small RNA sample decreases the background signals, as it filters out most of the RNA degradation products.This kit supports only single-indexed libraries at the moment (up two 48 barcodes are available). RNA samples can be suspended in RNase-free water or 1X TE buffer prepared with RNase-free water. RNA integrity should be assessed using the Agilent Bioanalyzer (or any similar system). For sample’s concentration, we prefer using fluorometric methods, such as Qubit or Ribogreen.
NGS DNA library preparation (Illumina compatible)
The KAPA Biosystems chemistry with in-house developed and validated adapters and indexing primers is used to prepare NGS DNA libraries for sequencing on any of the Illumina platforms. We refer to this system as “iTruS”. When multiplexing 24 or fewer libraries, we use the low throughput TruSeqLT to prepare 24 single-indexed libraries. For higher levels of multiplexing (more than 24 libraries), the high throughput (HT) iTruS is used to prepare dual-indexed libraries. The input DNA can be as low as 50 ng of high molecular weight DNA; however, we recommend submitting at least 300 ng of each sample in a maximum volume of 55 µl of TE. The DNA integrity is very critical for producing a library that captures the complexity of the genome. The DNA integrity should be checked on a 0.7% TAE Agarose gel before submission. DNA should be submitted in 1XTE.
NGS DNA PCR-free library preparation (Illumina compatible)
NGS DNA PCR-Free libraries eliminate PCR-induced biases. A minimum of 1 μg of input DNA in a maximum volume of 55 µl of TE is necessary to prepare a PCR-Free library.
Nextera XT DNA library preparation
The Nextera DNA sample preparation kit uses transposase to fragment the DNA and add adapters (known as tagmentation step) for single- or paired-end sequencing libraries. This kit is suitable for small genomes, such as prokaryotes, archae, and viruses. Also, it can be used to make libraries from PCR amplicons larger than 300 bp, plasmids, double-stranded cDNA, and concatenated amplicons. Up to 96 Nextera libraries can be multiplexed in the same sequencing lane/run. The main advantage of this kit is that it works with as low as 1 ng of starting DNA.
Please provide ≥20 µl of your ready to run library at a 10 ± 4 nM concentration in 10 mM Tris pH 8. If you would like to use a custom primer for your MiSeq run, please submit ≥80 µl at a concentration of 10 mM. Please add any QC information (Qubit data, Agilent run, fragment analyzer run, gel picture,…) available to the online order form.
SMARTer Pico cDNA Synthesis
Synthesis of cDNA from 1-2 ng of total RNA. Minimum total RNA concentration for this cDNA synthesis kit is 20pg/uL in a 50uL reverse transcription reaction.
Fragment Analyzer QC: Electropherogram Reading and interpretation
Fragment Analyzer QC: Electropherogram Reading and interpretation