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Universal Prokaryotic RNA-Seq Library Preparation Kit

Streamlined Prokaryotic RNA-seq library prep kit for strand-specific RNA-Seq library construction using between 100 ng to 500 ng of total RNA obtained from pure cultures or mixed populations of bacteria.




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Tab 01 / Overview
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Universal Prokaryotic RNA-Seq Library Preparation Kit

The Universal Prokaryotic RNA-Seq, Prokaryotic AnyDeplete® library preparation kit is an easy-to-use add and incubate system ideal for microbiological RNA sequencing studies. The Universal Prokaryotic RNA-Seq kit provides a complete solution for strand-specific RNA-Seq library construction using between 100 ng to 500 ng of total RNA obtained from pure cultures or mixed populations of bacteria. The integrated AnyDeplete technology enables rRNA depletion from a broad range of prokaryotes optimizing informative reads and reducing sequencing costs.

Sample types include purified RNA from bacterial cell lines, environmental samples, and microbiome studies.

Key Features of the Prokaryotic RNA-Seq library preparation kit include:

  • Complete solution for prokaryotic transcriptomics with a broad input range
  • Integrated rRNA depletion from a broad range of bacterial species with AnyDeplete technology
  • Compatible with samples with wide range of GC content
  • Pre-plated adaptors with unique indexes for each sample

Applications


  • Microbiome studies
  • Metagenomics studies
  • Whole transcriptome profiling
  • Gene expression
  • RNA sequencing

 




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Tab 02 / Highlights
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High quality data from a broad range of bacterial species

The Universal Prokaryotic RNA-Seq kit is designed to provide consistent data and performance across diverse bacterial communities or cell cultures.

  • Compatible across a wide range of % GC content
  • Efficient, integrated rRNA depletion from a broad range of prokaryotes
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Ribosomal RNA removal in bacteria

The composition of NGS libraries is determined by both the efficiency of depletion and the amount of rRNA initially present in the sample. For example, removing 90% of the rRNA from a sample containing an initial load of 98% rRNA and 2% other transcripts results in an NGS library with 17% non-rRNA (9.8% rRNA and 2% mRNA). When the same depletion rate is applied to a sample with 95% rRNA, the non-rRNA component will increase to 34%.

Sequencing alignment statistics for bacterial cDNA libraries

Data was generated with the Universal Prokaryotic RNA-Seq Library Systems or a random primer method, using 100 ng of total RNA input from the indicated sources. The resulting libraries were sequenced on the Illumina Genome Analyzer IIx using 4 libraries per lane (4-plex) with 40 bp single-read sequencing.

Efficient depletion of unwanted transcripts with AnyDeplete

AnyDeplete is a flexible solution for the removal of unwanted transcripts from your NGS libraries. Use the standard Prokaryotic AnyDeplete probe mix to remove rRNA from a broad range of bacterial species or customize the AnyDeplete probe mix for your model organism or mix probes together to target multiple species for host-pathogen studies.

  • Streamlined workflow to eliminate unwanted transcripts after library construction
  • Custom AnyDeplete probes available from our expanding list of organisms or design your own
  • Mix AnyDeplete probes together to target multiple transcripts and species

Custom targeted depletion of P. gingivalis rRNA reduces the percent of rRNA reads from 90% to 10% providing more useful information with the same number of sequenced reads.

 

Efficiently eliminate unwanted transcripts

AnyDeplete is able to efficiently eliminate rRNA reads from the library significantly reducing the number of uninformative reads, providing more useful data and lower sequencing costs

Validated AnyDeplete probes for mouse, drosophila and custom rRNA depletion

AnyDeplete efficiently reduces the amount of unwanted rRNA reads from standard mouse and Drosophila samples and can be customized to target unwanted reads from any organism such as P. gingivalis

Available AnyDeplete probes

Available custom AnyDeplete probes for Universal RNA-Seq with NuQuant library prep kit

Library quantification workflow with NuQuant

The NuQuant workflow for quantification of library molar concentration. Library molarity can be directly measured in less than 6 minutes using a Qubit with the appropriate NuQuant App or a standard fluorescent plate reader

NuQuant has the lowest variability for library quantification

A set of 8 libraries were distributed to 6 different users at multiple institutions. For each library and quantification method, the percent coefficient of variation (% CV) of molar concentration was calculated. NuQuant demonstrated the lowest variation across all users

Strong correlation between NuQuant molarity and read numbers

Sixteen libraries with varying insert sizes and concentrations were quantified with NuQuant. The libraries were then pooled by volume and sequenced. The graph shows the strong correlation between library molarity measured by NuQuant and the number of reads per sample demonstrating the accuracy of NuQuant




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Tab 03 / Specifications
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Specifications

Specification

Specification/Description

Compatible Platform

Compatible Platform

Illumina NovaSeq, HiSeq, MiSeq, NextSeq, MiniSeq

Starting Material

Starting Material

Total RNA isolated from bacterial sources

Input Amounts

Input Amounts

100 ng – 500 ng




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Tab 04 / Faq
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FAQ

What materials are provided with the Universal Prokaryotic RNA-Seq kit?

The Universal Prokaryotic RNA-Seq kit provides all necessary buffers, primers, enzymes and SPRI purification beads for generation of Illumina-compatible sequencing libraries. The kit also provides nuclease-free water for purification elution steps.

Does this system contain a SPIA®-based amplification?

No. The cDNA is generated with selective primers, but no SPIA-based amplification is used.

What equipment is required or will be useful?

Required equipment includes a microcentrifuge, pipettes, vortexer, a thermal cycler, a magnetic plate for 0.2 mL tubes, strips, or plates and a spectrophotometer or fluorometer. An Agilent Bioanalyzer or Fragment Analyzer may also be useful for optional analytical tests. A comprehensive list of required and recommended equipment can be found in Section II.B.

Can this system be used with other library preparation workflows?

The Universal Prokaryotic RNA-Seq kit is an end-to-end solution designed to generate libraries for Illumina sequencing starting from total RNA and have not been tested with alternative library preparation systems.

Can I use the Universal Prokaryotic RNA-Seq kit with RNA from any organism?

This system has been designed specifically for prokaryotes. Performance with other organisms may vary.

What methods do you recommend for RNA isolation?

We recommend a column-based method, including:

  • Norgen Biotek Total RNA, Soil RNA, or Stool RNA Purification Kits
  • Zymo Research QuickRNA™ Kit, ZR Fungal/Bacterial RNA MicroPrep™, or ZR Soil/Fecal RNA MicroPrep™
  • Arcturus PicoPure® RNA Isolation Kit
  • Ambion PureLink® RNA Mini Kit
  • Qiagen RNeasy Kits

Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.

Can I use TRIzol® or other phenol-chloroform based extractions for RNA isolation?

We do not recommend the use of TRIzol or similar methods as any carryover of organic solvents may inhibit downstream enzyme activity. If using TRIzol extracted RNA, we recommend using a column-based purification of the RNA prior to input into the kit.

Can I use carrier RNA during RNA isolation?

We do not recommend the use of carriers during RNA isolation. If a carrier is required, please contact Tecan NGS Technical Support for more information.

Will the use of RNA purification columns impact my data?

We have observed changes in alignment metrics and expression profiles with the use of purification columns such as the QIAGEN RNeasy column. We recommend consulting the manufacturer to ensure the RNAs of interest are retained after purification.

Do I need to use high-quality total RNA?

The Universal Prokaryotic RNA-Seq kits are designed to work with purified total RNA. When using purified total RNA, samples should be of high molecular weight with little or no evidence of degradation. While it is impossible to guarantee the highest levels of performance when using RNA of lower quality, this system should allow the successful analysis of somewhat degraded samples. With such samples, users may experience lower yields and may encounter affected sequencing metrics.

Do I need to perform an rRNA depletion or poly(A) enrichment step before processing samples with the Universal Prokaryotic RNA-Seq?

The system is designed to use total RNA as input. This system utilizes Tecan's customizable AnyDeplete technology to deplete targeted transcripts. rRNA depletion or poly(A) enrichment is not necessary.

How much total RNA do I need for amplification?

The selective priming process is designed to deplete rRNA from 100–500 ng total RNA input.

Can contaminating genomic DNA interfere with the Universal Prokaryotic RNA-Seq kit performance?

Yes, contaminating genomic DNA may be incorporated into libraries. For this reason we recommend DNase treatment during RNA purification. For an explanation of DNase requirements see section III.A. For DNase treatment of RNA samples, refer to Appendix A for guidelines.

How does your protocol enable strand retention?

The Universal Prokaryotic RNA-Seq kit utilizes targeted degradation of an incorporated modified nucleotide to ensure library inserts all carry the same directionality.

Does Tecan provide reagents for performing the fragmentation step of the protocol?

Tecan does not provide the reagents used in the fragmentation steps. We recommend using the Covaris instrument for cDNA fragmentation, as suggested in the “Materials” section of the product User Guide.

Please see section V.C. in the User Guide for recommendations for fragmentation with the Covaris instrument.

I don’t have access to a Covaris instrument. Can I use alternative fragmentation methods?

We have evaluated only Covaris fragmented DNA during the development of these systems. Other sonication instruments or enzymatic fragmentation may be suitable as long as the method generates a tight size distribution of cDNA fragments with a median size of 200–300 bp.

Can I combine the barcoded libraries prior to the PCR amplification step?

This is not recommended. The stoichiometry of barcoded libraries may be adversely affected by this modification to the workflow. We suggest that the libraries be amplified and quantified independently before being pooled for use on the sequencer.

Where can I safely stop in the protocol?

Samples can be placed in short-term storage at –20 °C after B. Second Strand Synthesis, after C. cDNA Fragmentation or after any of the bead purification steps.

What is the difference between RNAClean XP and AMPure XP SPRI beads? Can both be used interchangeably?

RNAClean XP beads are certified to be RNase and DNase free. We have tested both RNAClean XP and AMPure XP beads in our kits and observe no difference in performance between products.

What magnetic separation devices do you recommend for the SPRI bead purifications?

Due to the large number of commercially available magnets, we do not have a comprehensive list of compatible products. However, many magnets are compatible. As long as the magnet is strong enough to clear the solution of magnetic beads, it can be applied to the system. We have the following guidelines for selecting a magnetic separation device:

  1. Use a magnet designed for 0.2 mL tubes (PCR tubes), tube strips, or plates. Compared to magnets that are designed for 1.5 mL tubes, these minimize loss that can occur when samples are transferred from one tube to another.
  2. Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
  3. Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the Tecan workflow. This is also helpful to gain familiarity with the purification workflow.

How can I ensure maximum recovery of sample from the SPRI bead purification?

  1. Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
  2. Ensure that the beads are fully resuspended in solution before adding to the sample.
  3. Always use fresh ethanol during the washing steps. When preparing the ethanol, measure out the ethanol and water separately to ensure the desired ethanol concentration is obtained.
  4. Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.

How do I measure my final library yield? Can I use an Agilent Bioanalyzer to evaluate the product?

Please refer to section V.N of the User Guide for guidelines on quantitative and qualitative assessment. We recommend using a qPCR based-method in combination with the Agilent Bioanalyzer or Fragment Analyzer for the most accurate quantification.

How many bases do the Universal Prokaryotic RNA-Seq adaptors add to the library?

The adaptors add 124 bp to the library.

What sequencers are compatible with your libraries?

Universal Prokaryotic RNA-Seq libraries are compatible with Illumina sequencing platforms.

How much material should I load into the sequencer?

Please follow manufacturer’s recommendations for library QC, quantification, balancing and loading of the amplified library on the sequencer.

What kind of sequencing primers can I use with your libraries?

The Universal Prokaryotic RNA-Seq kits are designed for use with the standard Illumina sequencing primers for both single- and paired-end sequencing.

Can the Universal Prokaryotic RNA-Seq kit be used with paired-end sequencing?

Yes, they can be used for both single- and paired-end sequencing. Special consideration should be given to the expected insert size in the paired-end assay.

What kind of error correction is used to minimize the impact of sequencing errors in the barcodes?

For experiments using the Universal Prokaryotic RNA-Seq, please follow the Illumina recommendations on parsing barcodes. The sequences of the Tecan barcodes must be input prior to parsing.

Can I use standard alignment algorithms to analyze strand-specific sequencing data?

Yes. Strand-specific reads can be processed and mapped to reference sequences using the same methods used for other RNA-Seq libraries. Note that in libraries generated by the Universal Prokaryotic RNA-Seq, the forward read corresponds to the sense strand.

Will the presence of extrachromosomal material in total RNA impact my data?

It is possible to see a higher proportion of unmapped reads in the context of some bacterial strains with extrachromosomal content, such as plasmids.




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Tab 05 / Literature
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Literature

Type
Title
No
User Guide
M01502 V1
Product Sheet
401100 V1.1
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