Our Services in the Area of NextGen Sequencing

• DNA Preparation   
• Library Preparation 454  
• Library Preparation SOLiD  
• Multiplexing & Pooling of the Samples
  
• Libraries Overview
  
• Sequencing 
  
• Bioinformatics  
  

DNA Quality Control

For every project Microsynth performs a quality control of the input DNA. Depending on the project, this is done by agarose gel electrophoresis, Agilent bioanalyzer electrophoresis or Sanger sequencing reactions.

BAC DNA Isolation

In contrast to Sanger sequencing technology, in a BAC sequencing project a possible contamination with E. coli DNA cannot be excluded and may result in a loss of total capacity. Microsynth has established an own protocol for the isolation of BAC DNA prior to sequencing, achieving a low E. coli DNA content.

Shotgun & Paired-end

Microsynth suggests either shotgun library or 3kb or 8kb long-tag paired-end library for 454 technology. The use of the shotgun library preparation produces reads with read-length range from 500 to 1000  bases, which are assembled with the GS De Novo assembler (or the GS Reference Mapper) in long contigs.

The use of the 3 kb or 8kb long-tag, paired-end protocol allows the sequencing of two reads from each end of a 3'000 or 8'000 base span. With this approach, a scaffolding of the genome is provided.

Fusion Primers for Amplicon Sequencing

The procedure for preparing a DNA sample for GS FLX amplicon sequencing consists of a simple PCR amplification, but requires special "fusion primers" which must be designed by the user according to the specific requirements of the experiment. Fusion primers have the following characteristics:

Two types of kits can be used for amplicon sequencing. The typical Lib-A kit is used for bidirectional sequencing as it contains both the A and B capture beads and for unidirectional sequencing when using either A or B capture beads. However an alternative approach for unidirectional sequencing is the use of the Lib-L kit. In our laboratory we did compare the two kits for unidirectional sequencing: Lib-A versus Lib-L and do recommend the use of the Lib-L kit for amplicon unidirectional sequencing. The design of the fusion primers depends on the kit used.

Bidirectional amplicon sequencing

The fusion primers must contain the GS FLX Titanium adaptor A and adaptor B of the kit Lib-A. It includes also a four base library key sequence (TCAG) which is always the same, as dictated by the requirements of the GS FLX Genome Sequencer System, an optional Multiplex Identifier (MID) sequence to allow further sorting of the samples (in case of amplicon pooling) and a sample specific sequence. 

When using Lib-A, the sequences of both forward and reverse primers are as follows:

Forward primer A:

5’ CGTATCGCCTCCCTCGCGCCATCAG-MID-template-specific-sequence-3'

Reverse primer B:

5’ CTATGCGCCTTGCCAGCCCGCTCAG-MID-template-specific-sequence-3'

Unidirectional amplicon sequencing

The fusion primers must contain the GS FLX Titanium adaptor A and adaptor B of the kit Lib-L. As the sequencing will be unidirectional, the sequencing will be made from adaptor A only. An optional Multiplex Identifier (MID) sequence to allow further sorting of the samples (in case of amplicon pooling) will be add only on the forward (adaptor A). The four base library key sequence (TCAG) is also present as well as a sample specific sequence. Even for unidirectional sequencing a reverse primer B is necessary for attachment on the capture bead B but it will not contain any MIDs.

When using Lib-L, the sequences of both forward and reverse primers are as follows:

Forward primer A:

5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG-MID-template-specific-sequence-3'

Reverse primer B:

5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-template-specific-sequence-3'

• It isvery important to use the kits Lib-A or Lib-L corresponding to their respective adaptors as those have different sequences
• Microsynth  provides high quality purified fusion primers for further 454 sequencing
• When you design your project, please consider the following:
  • Primer quality: the better the better …
  • Uniformity of fragment length: the closer the better
  • Range of fragment length: 200 – 800 bp (300 – 500 bp would be ideal)

RNA Sequencing: cDNA Preparation for Transcriptome Projects

Microsynth provides normalized or non-normalized cDNA libraries. The main application of 454 technology in RNA sequencing is the production of reference transcriptomes using normalized cDNA.

Shotgun, Paired-end & Mate-paired*

Microsynth suggests either a fragment library or mate-paired library for SOLiD technology. Fragment sequencing produces reads up to 75 bases (or 75 + 35 if SOLiD paired-end chemistry is applied). If desired and if sufficient, it is possible to produce shorter reads only (35 bases). Mate-paired sequencing produces reads of 60 + 60 bases which are 1-6 kb apart.

* Please note that 454 paired end is not equal to SOLiD paired end!

RNA Sequencing: cDNA Preparation for Transcriptome Projects

Libraries preparation and multiplexing for either whole transcriptome or small RNA are provided for SOLiD technology.

DNA Enrichment

Using Agilent SureSelect technology, Microsynth provides DNA enrichment either for whole human exome or for customized targeted enrichments.

In order to increase the efficiency of the run, Microsynth labels the different DNA libraries with barcodes. The subsequent pooling is based on qPCR values.         

Microsynth performs the sequencing run on either a full run or a partial run according to the project. The repartition on the run is optimized for the customers' needs.

On the Roche FLX GS system, we provide full run (700 Mb), 1/2 (350 Mb),1/4 (120 Mb) and 1/16 (17 Mb) runs.

In order to avoid long delivery times for small projects and to allow the customers to plan in advance, partial runs (16/16) are processed on a regular basis.

Please note:

• If you want to be informed about partial runs and be regularly advised about the next run, contact us at genome@microsynth.ch

On the SOLiD 5500xl, the runs are divided in 2 FlowChips, and each of these is further divided in 6 independent lanes. Each sample can be run separately, and the customer's projects are optimally divided on the different lanes. The read length can be optimized from 35 up to 75 bases. For example, a small RNA project can be processed with only 35 bases read length.

The use of the supplementary ECC (exact call chemistry) module guaranties high accuracy even in the absence of a reference sequence. The total capacity per run is max 180 Gb.

Delivery of the Results

The sequencing data are delivered per HTTPS download or on a hard drive.

Microsynth Offers the Following Bioinformatic Services:

• 454 raw data (FASTA format, Quality Scores, Sff files)
• SOLiD raw datas (csfasta format, Quality scores)
• De novo assembly
• Mapping against a genome reference 
• Mapping against a transcriptome reference 
• SNP analyses
• Sorting of the reads according to theirs tags
• Copy number variation analyse
• Gene expression quantification
• Removal of contaminant sequence
• Free one-month trial license for the software Genomic Workbench from CLC bio