Microsynth can propose the following applications using the FLX sequencing technology
DNA Quality control
Microsynth performs for every project 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.
Whole genome sequencing: De Novo and resequencing
Microsynth propose both shotgun reads and or 3kb, 8kb and 20 kb long-tag paired end reads.
- The use of the shotgun library preparation produces reads with read length range from 300 up to 500 bases which are assembled with the GS de Novo assembler (or the GS reference Mapper) in long contigs.
- The use of the 3 kb, 8kb or 20kb Long-Tag paired end protocol allows the sequencing of approximately 150 bases from each end of a 3'000, 8'000 or 20'000 base-span. The contigs obtained are more numerous (see table 1) but with this approach a scaffolding of the genome is provided.
Microsynth prepares the DNA for the FLX run according to one of the two options mentioned above, performs the sequencing run on either a full large PTP (picotiterplate), on a 1/2, 1/4, 1/8 or 1/16 PTP.
An electronical assembling is made with the software "GS de Novo Assembler" and or " GS reference Mapper".
The sequencing data will be delivered on a DVD. The data consist in Raw Reads (FASTA format, Quality Scores, Sff files) and the Assembly data (Fasta formats and Ace files)
Advantages of the FLX sequencing system in whole genome sequencing
In resequencing or comparative genomics projects, large discrepancies between the sequenced genome and the reference genome can happen and make any alignment impossible. The long reads (400 bases) of the FLX Titanium system give the unique advantage, to allow also an assembling as a de Novo project by using the "GS de Novo Assembler" and to obtain by this way usable datas.
Microsynth also provides a library generation service for Sanger sequencing. If desired a hybrid approach of Sanger sequencing and 454 sequencing can be performed to profit from the advantages of both technologies.
Example of de Novo assembly results
|
Organism 1 De Novo Shotgun |
Organism 1 De Novo Paired end |
Organism 2 De Novo Shotgun |
Total of bases |
153.4 Mb |
115.6 Mb |
133,7 Mb |
Total of reads |
607’102 |
694’566 |
572’232 |
Large contigs (>500bp) |
|
|
|
Nb of large contigs |
82 |
305 |
87 |
Largest Contig |
158.4 kb |
59 kb |
421.4 kb |
Avg contig size |
33,7 kb |
9 kb |
57 kb |
N50 contig size |
60.7kb |
17.4 kb |
226.2 kb |
Nb of bases |
2,763 Mb |
2,762 Mb |
4,971 Mb |
All contigs |
|
|
|
Total nb. of contigs |
141 |
963 |
141 |
Total Nb of bases |
2.777 Mb |
2.883 Mb |
4,984 Mb |
Scaffold |
- |
- |
- |
Nb. of scaffolds |
- |
16 |
- |
Nb. of bases |
- |
2,765 Mb |
- |
Largest Scaffold |
- |
1.555Mb |
- |
Avg. Scaffold size |
- |
172.8 kb |
- |
N50 Scaffold size |
- |
1.555 Mb |
- |
Organism. 1 : Clostridium chauvoei – with the courtesy of Prof. J. Frey, Bakteriologie, Fakultaet, Universität Bern
Organism 2 : Escherichia coli strain – with the courtesy of Prof. R. Stephan, Institut für Lebensmittelsicherheit & Hygiene, Universität Zürich
BACs sequencing
Depending on their sizes several BACs can be sequenced during one run. Two options can be used:
- The PTP is divided physically in 2, 4, 8 or 16 regions by the use of a gasket.
- Multiplex Identifiers (MIDs) containing a unique nucleotide sequence are ligated to the samples during the libray preparation and are used to tag the samples. The samples can then be pooled together.
The preparation of the BAC-DNA libraries for the FLX , the sequencing runs, the electronical assembling and the transmission of the datas are similar to what is described above in the whole genome sequencing section.
It is important to be aware of the fact that, unlike with Sanger sequencing technology, by BAC sequencing a possible contamination with E. coli DNA cannot be excluded and may result in a loss of total capacity.
cDNA sequencing
cDNA for direct sequencing or RNA for cDNA libraries preparation (cloning of full-length cDNA, cloning of small non-coding RNA) and subsequent sequencing can be sent to Microsynth.
- < 600 bases : the DNA fragments are treated like amplicons
- >2000 bases : the DNA fragments have to be fractionated by a shotgun procedure
The preparation of the cDNA for the FLX , the sequencing runs and the transmission of the datas are similar to what is described above in the whole genome sequencing section.
Electronical assembling : If de Novo, the alignment is done electronically with the software "GS de Novo Assembler".If a reference sequence exist, the alignment is done electronically with the software "GS Reference Mapper"
Amplicon Sequencing
The procedure for preparing a DNA sample for 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.
The Fusion Primers must contain the GS FLX Titanium Primer A and Primer B (which includes a four base library key sequence TCAG) , are always the same, as dictated by the requirements of the GS FLX Genome Sequencer System. An optional Multiplex Identifier (MID) sequence may be added to allow further sorting of the samples.
They have the following characteristics:
Forward primer (Primer A)
I. Primer A: 5’ CGTATCGCCTCCCTCGCGCCATCAG-MID-template-specific-sequence-3'
II. Primer B: 5’ CTATGCGCCTTGCCAGCCCGCTCAG-MID-template-specific-sequence-3'
The 3’-part of each Fusion Primer is specific to each Amplicon and is used by the Amplicon Variant Analyser software to assign reads:
The amplicons are pooled (equimolar pooling) in 1, 1/2, 1/4, 1/8 or 1/16 run. The number of amplicons that can be pooled together is a compromise between ensuring a high probability of finding a variant and pooling the largest possible number of amplicons.
As an example : if a desired sentivity for a variant within a sample is 10%, 5% or 2%, ans 50 reads pro amplicons, the number of amplicons that can be pooled in 1/16 of a PTP plate (30'000 reads) will be respectively 60, 30 and 12.
The sequencing runs and the transmission of the data are similar to what is described above in the whole genome sequencing section.
Electronical analysis is made with the software « GS Amplicon Variant Analyser software »
Example of deep sequencing of PCR products
To pool several samples within one region the PCR products were barcoded by fusionprimers. Six different fusionprimers with a 6 bp barcode were used to deep sequence totally 36 samples on six regions.
Sample | Region | Barcode | Reads per region | Reads per sample |
1 | 1 | 1 | 9476 | 1688 |
2 | 2 | 1866 | ||
3 | 3 | 1220 | ||
4 | 4 | 1479 | ||
5 | 5 | 1293 | ||
6 | 6 | 1925 | ||
7 | 2 | 1 | 11769 | 2171 |
8 | 2 | 2626 | ||
9 | 3 | 1276 | ||
10 | 4 | 1321 | ||
11 | 5 | 2571 | ||
12 | 6 | 1800 | ||
13 | 3 | 1 | 9444 | 1887 |
14 | 2 | 1463 | ||
15 | 3 | 1165 | ||
16 | 4 | 1840 | ||
17 | 5 | 1494 | ||
18 | 6 | 1599 | ||
19 | 4 | 1 | 8465 | 1263 |
20 | 2 | 1984 | ||
21 | 3 | 1177 | ||
22 | 4 | 1433 | ||
23 | 5 | 1337 | ||
24 | 6 | 1268 | ||
25 | 5 | 1 | 11892 | 2614 |
26 | 2 | 2161 | ||
27 | 3 | 1947 | ||
28 | 4 | 2039 | ||
29 | 5 | 1297 | ||
30 | 6 | 1855 | ||
31 | 6 | 1 | 8734 | 1388 |
32 | 2 | 1394 | ||
33 | 3 | 1440 | ||
34 | 4 | 2127 | ||
35 | 5 | 992 | ||
36 | 6 | 1401 |
Table1: Number of reads received per region and sample after equimolar pooling of the PCR products. With the courtesy of Prof I. Roditi, Institute of Cell Biology, University of Bern
Metagenomics
The long reads obtained with the Titanium series enables diversity, metabolic and microbial abundance studies of the metagenomes of interest.
Two approaches are possible for metagenomic projects:
- Analysis of the 16sRNA of a community. This approach is equivalent to an Amplicon project.
- Shotgun sequencing of the whole community DNA.
