Fasteris SA

De novo sequencing is a Whole-Genome Sequencing (WGS) method that allows sequence determination of a novel genome with high resolution and accuracy. It is suitable for analysis of genomes of any size and complexity.
De novo sequencing approach differs from the genome re-sequencing method by the library construction setup. In addition to shotgun libraries it is highly recommended making use of libraries derived from larger genomic fragments. This approach will significantly improve the quality of the assembly and the average scaffold size of the derived contigs.
For organisms without reference (de novo) Fasteris offers a combination of following library preparation protocols:

• Fasteris de novo DNA in house developed protocol for genomes without reference. The protocol was designed to facilitate genomic assembly and has 2 main advantages:
  
  • selection of precise insert size by gel size selection
    
  • minimization of chimeric effect
    
    • Requirements: 1-2 µg of genomic DNA 50 µL DNase-free water.
      
    • NB: Alternately shotgun Nano DNA libraries of various sizes can be used.
      
• Nextera Mate-Pair protocol produces long-insert paired-end libraries that are used for generation of large genomic scaffolds. For preparation of this type of library high quality chromosomal DNA is needed
  
  • Requirements: 4 µg of genomic DNA 50 µL DNase-free water.
    
    • NB: Mate-Pair libraries are available with a jumping distance of 3 kb (default size) and up to 5 kb (optional).
      

NB: For de novo sequencing approach we recommend aiming at least 50-fold to 70-fold genome coverage.

NGS sequencing options:

Fasteris offers sequencing on Illumina HiSeq 2500, HiSeq 4000 and MiSeq platforms, coverage can be adjusted to your needs.

Transcriptome sequencing (RNA-seq) is a Next-Generation Sequencing method that enables to explore the complete set of transcripts of living cells of model and non-model organisms. Such investigation is essential for interpreting the unique functional organism status in normal physiological conditions or diseases, in response to various stimuli or at different developmental stages.
Transcriptome sequencing generates millions of strand-orientated reads derived from mRNA or/and non-coding RNA populations converted into cDNA. The method allows not only precise and accurate quantification of gene expression, but also identification of novel transcripts and isoforms (i.e. alternatively-spliced transcripts).
Fasteris offers a wide range of protocols to help finding the answer to your biological questions:

• Standard stranded mRNA protocol includes poly-adenylated transcripts selection, followed by cDNA library construction using illumina TruSeq Stranded mRNA Library Prep kit.
  
  • Requirements: 500 ng to 1 µg of total RNA in 20 µL RNase-free water.
    
• RiboZero stranded RNA protocol includes species-specific ribosomal RNAs depletion, followed by cDNA library construction using illumina TruSeq Stranded RNA Library Prep kit. The protocol is recommended for whole-transcriptome assessment across coding and non-coding RNAs.
  
  • Requirements: 1 µg of total RNA in max. 20 µL RNase-free water.
    
  • NB: Before opting for RiboZero protocol, please check if your organism is among the listed species for which Illumina ensures RiboZero kit compatibility (www.illumina.com/products/rrna-removal-kit-species-compatibility.html).
    RiboZero stranded RNA protocol can also be adjusted for low amount of starting material (e.g. 50-100 ng of total RNA in max. 10 µL RNase-free water).
    
• DSN treatment (using Duplex-Specific Nuclease) for degraded samples, transcriptome discovery or nascent RNA studies. After preparation of the library on total RNA or poly-A selected transcripts, a DSN treatment is performed in order to reduce the prevalence of the most abundant cDNA molecules derived from highly expressed transcripts, including rRNA.
  
  • NB: This approach is not suitable for accurate transcripts quantification.
    

Please note: None of the above three protocols is adapted or suitable for transcripts smaller than 100 bp. Thus, if you are looking for miRNA expression, please consider the small RNA protocol. In addition, it is essential to perform DNase treatment prior to library preparation. It is strongly recommended to perform it during the RNA extraction procedure.

NGS sequencing options:

Fasteris offers sequencing on Illumina HiSeq 2500, HiSeq 4000 and MiSeq platforms, with up to 96 libraries multiplexed per lane. Coverage can be adjusted to your needs.

Small RNA sequencing (small RNA-seq) is a Next-Generation Sequencing method allowing to explore small non-coding RNAs (18-35 nt long). Small RNA molecules are the powerful mediators, regulating gene expression at the transcriptional, post-transcriptional and translational levels in organisms of different complexity. Small RNAs play an important role in organism functional regulation in normal physiological conditions (cell proliferation, apoptosis) or diseases, such as cancer. Small RNA-seq also allows analysing virus and infected host (such as plants) interactions and even reconstructing viral genome.
The three major well-known and well-described small RNA classes are: microRNAs (miRNAs), interfering RNAs (siRNAs) and Piwi-associated RNAs (piRNAs). Small RNA-seq allows to examine the differential expression of all these 3 small RNA classes and also to discover novel small RNAs.
For small RNA studies Fasteris offers the following protocols:

• The standard small RNA protocol is based on the illumina TruSeq small RNA kit. Starting from total RNA we perform polyacrylamide gel to select small RNAs (including miRNAs, siRNAs and piRNAs) with 18-30 nt fragment size before the library preparation. For Mammalian samples beads size selection can be performed instead of polyacrylamide gel. The protocol is suitable not only for differential expression analysis, but also for identification of novel small RNAs and for virus-derived small RNA in infected species.
  
  • Requirements: 2-3 µg of total RNA in 10 µL RNase-free water or 50-100 ng of IP RNA in 6 µL RNase-free water.
    
• Anti 2S treatment using highly specific probes developed in house to deplete 30 nt long 2S RNA from Drosophila organism and overcome the contamination of this highly abundant rRNA in the final library.
  
  • Requirements: 3 µg of total RNA in 10 µL RNase-free water.
    
• Treatment for non 5’ monophosphate small RNAs. The procedure is useful for 5’-cap and 5’triphosphate (5’-ppp) RNAs. Several consecutive enzymatic treatments ensure 5’-monophosphate state of all RNAs which is essential for adapter ligation and inclusion into library composition.
  
  • Requirements: 3 µg of total RNA in 10 µL RNase-free water.
    
• NB: For small RNA protocol the final data is provided with the adapter trimmed and separated by size sequences by default! These additional treatments aim to facilitate the following bioinformatics analysis.
  

NGS sequencing options:

Fasteris offers sequencing on Illumina HiSeq 2500, HiSeq 4000 and MiSeq platforms, coverage can be adjusted to your needs.

Chromatin immunoprecipitation (ChIP) is a method used for detection of interactions between protein and DNA in living cells. Next-Generation Sequencing of the derived and purified DNA allows the identification of genomic regions associated with specific proteins even if the sequence of the organism is unknown. The approach is used for identification of DNA binding sites of transcription factors, histone localization, etc.
ChIP-seq library preparation is performed with illumina TruSeq ChIP kit and up to 24 samples can be multiplexed in the sequencing lane.

• Requirements: 20 ng of ChIP DNA in 30 µL water (fragments ranging 100-500 bp).
  
• NB: For fragments that are above 600 bp an additional fragmentation step can be performed at Fasteris.
  
• NB: Please note that the Bioanalyzer profiles or gel pictures of the samples should be provided by email.
  
• NB: We recommend using an input sample as a control to help remove noise and allow more accurate peak detection.
  

NGS sequencing options:

Fasteris offers sequencing on Illumina HiSeq 2500, HiSeq 4000 and MiSeq platforms, coverage can be adjusted to your needs.

Transposon sequencing (Tn-seq) is a Next-Generation Sequencing (NGS) method for genome-wide scale profiling allowing accurate detection of transposon insertions sites.
Most commonly Tn-seq is used for the analysis of microorganism mutant pools to identify genetic aspects associated with certain phenotypes or to get insight to genetic basis required for fitness; for example such as antibiotic resistance gene identification by comparison of the mutant abundance after the growth in the selective conditions.
Fasteris in house developed Tn-seq protocol can be adjusted to your experimental needs, depending on the transposon used.
- Requirements: 3 µg of genomic DNA in maximal volume of 50 µL water.
- NB: We will need to know which transposon (including exact sequence) was used in your experimental procedure.

We use the highest quality Sanger (Capillary) DNA Sequencing methods, personalized advice and next day results.
From Economy to Full service or from Ready-to-Run to Customized service, we offer the most flexible solutions to suit your needs.
We are dedicated to providing the highest quality sequences from any sample.

We have one of the longest and most extensive experience as service provider with the Illumina platform.
We provide unparalleled customer support, not only with experimental design, library preparation options and sequencing methods, but also with bioinformatics analyses.
Our combined expertise is particularly useful for difficult projects that require innovative solutions, but also quite valuable for new customers.
We strive to provide biologically relevant information by using a variety of tools to visualize and interpret the results.
We are always ready to discuss innovative ideas and help you achieve the optimal results.