Genome Sequencing

Eurofins Genomics India deals with a variety of comprehensive services for genomes form prokaryotes to Eukaryotes. A complete and accurate sequencing of entire genome for organism for better understanding of genomic structural variations and diversity among species. We offer complete packages of each services including sample preparation, sequencing and Bioinformatic analysis to provide the optimum sequencing strategy. The different application ranges are listed below.

De novo genome sequencing:

• Sequencing of unknown genomes (no reference sequence available in public database)
• The de novo strategy is recommended, if large genomic re-arrangements or InDels has accumulated in the genome.
• Assembly of shotgun reads into contigs based on sequence identities. Contigs are oriented and ordered by or Mate pair / long jumping distance (LJD) libraries

Re-sequencing projects

• Ideal when a good reference sequence is available
• Mapping of shotgun reads to the reference sequence, optional variance analysis
• We are offering genome sequencing service for the following areas

De Novo Genome Sequencing

De novo Sequencing refers to sequencing of a novel or unique genome in the absence of a reference sequence available for alignment in public databases. It is mainly the combination of sequencing, assembly of reads, genome annotation, functional annotation and also advanced bioinformatic analysis such as comparative genomics analysis, etc. In contrast to traditional PCR/Sanger techniques, Next Generation Sequencing approach multiplex a number of samples, providing identifications of SNP/InDel/CNV (copy number variation)/SV (Structural variation) with great consistency, turnaround time and cost efficacy. NGS technologies comprising, NextSeq500, NovaSeq6000, HiSeq2500, PacBio and Nanopore have widely extend their applications in animal/microbe/plant genome de novo sequencing.

Advantages:

• Generates precise reference sequences, even for complex or polyploid genomes
• Information can be generated for mapping genomes of novel organisms or finishing genomes of known organisms
• Illuminates highly similar or repetitive regions for accurate de novo assembly
• Categorizes structural variants and complex rearrangements, such as deletions, inversions, or translocations

Bacterial and Fungal Genome Sequencing

The gold standard for de novo sequencing of small genomes combines backbone sequencing (shotgun-libraries) and scaffolding using paired-end libraries with large jumping distances. By strategically combining sequencing and scaffolding of up to 5 libraries, we get the longest average scaffold sizes and smallest number of remaining gaps. This strategy drastically reduces your post-sequencing efforts by cutting down on gap closing and manual editing time.

The appropriate layout of different Mate pair / LJD libraries as well as the sequencing technology depends mainly on the complexity & size of your genome. We would be happy to advise you to find the most convenient strategy.

Methodology:

We accept NGS grade high quality gDNA, Pure Microbial cultures (non‐pathogenic) and Fungal mycelia

Sample Requirement:

• Tissue: minimum 30-100mg of fungal mat or mycelia with or without PBS should be shipped in dry ice to the Eurofins facility.
• Genomic DNA: 2.0-3.0µg of NGS grade intact double stranded genomic DNA, free from RNA and protease contamination dissolved in Nuclease free water or 1x TE buffer should be shipped in cool pack to the Eurofins India facility. gDNA should have an Absorbance ratio (A_260/280) of 1.8-2.0.
• Culture: Pure isolated bacterial or fungal culture (minimum 2 x 10⁷cells in duplicate) should be shipped in cool pack to Eurofins facility.

Sample Quality Control:

• Pure high quality gDNA will be isolated from the received tissues or cultures using commercially available validated kits.
• The quantification and qualification of gDNA will be done on agarose gel electrophoresis and NanoDrop/Qubit Fluorometer analysis, respectively.
• DIN value estimation will be carried out by Agilent 4200 Tape Station.

Library Preparation:

• Small fungi and bacterial genome: Achieved by combining both paired-end shot gun library with a long jumping distance library/Mate pair library.
• Large fungal genome: Achieved by combining 3-4 paired-end shot gun libraries with 2 long jumping distance library/Mate pair library.
• The quality of prepared library will be authenticated on Agilent 4200 Tape Station.

Sequencing:

• Sequencing on Illumina NextSeq500 platform with 2 x 150 bp v2 chemistry or HiSeq 2500 with 2 x 125 bp v4 chemistry or NovaSeq with 2x150 bp chemistry.

Deliverables :

• Raw data will be available for download as a compressed archive of FASTQ files for each sample.
• Comprehensive compiled report and data set will be shared in link or Pen Drive or Hard Disk.
• For analysis please visit Bioinformatics Services

Turnaround time

• Provided that there are no unexpected difficulties (biological or technical), 6-8 weeks after successful clearance of QC step. It depends upon data size, scope, technology selected, number of samples and complexity of the project.
  
  
  

Eukaryotic Genome Sequencing

Rely on wide-ranging and enormous experience of Eurofins Genomics for de novo sequencing of large eukaryotes.

Due to tremendous up-gradation of sequencing platform and process, the prices / bp dropping eukaryotic genome sequencing projects are rapidly being implemented in projects worldwide. Eurofins Genomics has extended profound experience in de novo sequencing and assembly of the larger, more complex, and repeat-rich genomes of higher eukaryotes. Our expertise ranges from insects to large plants. One of the big challenges of such projects is the scaffolding of the contigs from shotgun sequencing. That is why Eurofins Genomics focused on developing paired-end libraries with large jumping distances for sequencing by the NovaSeq, Illumina HiSeq 2500 or NextSeq 500. These unique long jumping distance (LJD) libraries or Mate pair libraries offer ultra-high throughput and cost-efficient scaffolding of contigs.

The appropriate layout of different Mate pair / LJD libraries as well as the sequencing technology depends mainly on the complexity & size of the eukaryotic genome.

Based on the project requirement, our scientific expert team would be happy to advise you about the most convenient strategy. The one of the best strategy to achieve optimum high throughput sequencing data would be

• The type of long jumping distance (LJD) or Mate pair libraries combined with the shotgun library of insert sizes of 3-5 kbp and 8-10 kbp.
• Optional, included cDNA library sequencing in order to support gene annotation or take advantage of sequencing specialized libraries, like for example methylation filtered libraries that support coverage of transcriptional active euchromatic parts of the genome.

Methodology:

We accept NGS grade high quality gDNA, tissue samples for this Service.

Sample Requirement:

• Tissue: minimum 3-5 gm of tissue should be dissect aseptically and fast frozen in liquid nitrogen or in RNA later shipped in dry ice to Eurofins facility.
• gDNA: Submit 3-5mg of RNA-free genomic DNA (NGS grade) of molecular weight >40kb, Nanodrop A260_/280 ratio >1.8; A_260/230=2.0-2.2; and at a concentration of ~50 ng/ml (dissolve in low TE buffer).

Sample Quality Control:

• RNA free NGS grade gDNA isolation will be carried out using commercially available DNA isolation kit.
• The qualification and quantification of gDNA will be attained by agarose gel electrophoresis and NanoDrop/Qubit Fluorometer analysis, respectively.
• DIN value estimation will be carried out by Agilent 4200 Tape Station.

Library Preparation:

• Genome coverage can be achieved by combining 3-4 paired-end shot gun libraries with 2-4 long jumping distance library / Mate pair library with a bridge gap of 3-5kbp and 8-10 kbp.
• Prepared libraries QC will be done using Agilent 4200 Tape Station.

Sequencing:

• Sequencing on Illumina NextSeq500 platform with 2 x 150 bp v2 chemistry or HiSeq 2500 with 2 x 125 bp v4 chemistry or NovaSeq with 2x150 bp chemistry.

Deliverables:

• Raw data will be available for download as a compressed archive of FASTQ files for each sample.
• Comprehensive compiled report and data set will be shared in link or Pen Drive or Hard Disk.
• For analysis please visit Bioinformatics Services

Turnaround time

Provided that there are no unexpected difficulties (biological or technical), 6-8 weeks after successful clearance of QC step. It depends upon data size, scope, technology selected, number of samples and complexity of the project

BAC, Fosmid, Plasmids and Virus Genome Sequences

Eurofins Genomic India provides a cost-efficient and advanced way for sequencing of small genomes and large insert constructs irrespective of the types.

Achieve high quality de novo sequencing of small genomes and constructs by sequencing your samples using a long read technology. Easily bridge small repetitive elements and benefit from longer contigs.

The successful completion of many projects built our expertise in sequencing of all kinds of small genomes and insert constructs like:

• BACs, PACs, Cosmids and fosmids
• Plasmids and phages
• Mitochondria and chloroplast samples
• DNA- and RNA-viruses

Methodology:

We accept NGS grade high quality gDNA, fosmids, plasmids and viral nucleic acids.

Sample Requirement:

• Submit of high molecular weight, NGS grade 2-5 mg of RNA-free DNA/plasmids/cosmids, NanoDrop A260/280 ratio >1.8; A260/230=2.0-2.2; and at a concentration of ~50 ng/m
• If viral RNA, NanoDrop A_260/280 ratio >1.9 or above; A_260/230=2.0-2.2; and at a concentration of ~50-100 ng/m

Sample Quality Control:

• The qualification and quantification of gDNA will be carried out by agarose gel electrophoresis and NanoDrop/Qubit Fluorometer analysis, respectively.
• Quality check of viral RNA will be carried out using NanoDrop/Qubit Fluorometer analysis.
• DIN value estimation of gDNA will be carried out by Agilent 4200 Tape Station.

Library Preparation:

• Library of small genome will be achieved by using Nextera XT DNA library preparation kit or TruSeq Nano DNA library preparation kit from illumina.
• Viral genome library will be prepared using in-house developed protocol.
• Prepared libraries QC will be carried out using Agilent 4200 Tape Station.

Sequencing:

• Sequencing on Illumina MiSeq platform using long read paired-end chemistry of 2 x 300 bp.
• Viral libraries will be sequenced on NextSeq500 platform using 2 x 150bp v2 chemistry or NovaSeq using 2x150bp chemistry.

Deliverables:

• Raw data will be available for download as a compressed archive of FASTQ files for each sample.
• Mapping on the reference genome
• Alignment summary statistics,
• Gene annotation
• SNP Discovery
• Comprehensive compiled report and data set will be shared in link or Pen Drive or Hard Disk.

Turnaround time

6-8 weeks after clearance of QC process. It depends upon data size, scope, technology selected, number of samples and complexity of the project. The TAT is offered expecting no biological or technical difficulties for processing of all project samples.

 

Re-sequencing of Genomes

Identify the differences with Illumina NovaSeq, HiSeq 2500 or Illumina NextSeq 500 or Illumina MiSeq technology.

Re-sequence whole genomes and identify genetic variations, single base mutations, insertions and deletions compared to the reference genome. Based on your study's aims and the size of the genome, Eurofins Genomics expert team will address your re-sequencing project using suitable sequencing platform either NovaSeq, Sequel, HiSeq 2500 or NextSeq500 or MiSeq technology.

Re-sequencing with NovaSeq6000

Due to huge output sequencing capacity, Novaseq is one of the trending sequencing platform accepted worldwide for genome sequencing studies. The Novaseq is ideal for the large whole genome sequencing with denovo as well referenced based study. The platform get popularity due to accumulation of various samples depend on the data output with 4 different type of flow cells with combination of different diverse library. Eurofins offer big genome sequencing project on our Novaseq platform.

Re-sequencing with Illumina HiSeq 2500

The HiSeq 2500 is ideal for whole genome re-sequencing of large eukaryotic genomes with well-known references. The tremendous data outputs allow sequencing of 6 human genomes per run or tens of fungal genomes per channel.

Re-sequencing with Illumina NextSeq500

The NextSeq 500 is ideal for whole genome re-sequencing of large eukaryotic genomes with well-known references. The fast, integrated, sample-to-results workflow enables many sequencing applications—including transcriptomes, exomes, and targeted panels—in a single run.

Resequencing with Illumina MiSeq

With reads of up to 300 bp, the MiSeq sequences up to 24 bacterial genomes in one single run. Delivering turnaround times of only 3-4 weeks for such projects is a walk in the park for the Illumina MiSeq. If you are looking for high quality and fast re-sequencing of bacterial genomes, the MiSeq is the technology of choice.

Re-sequencing project specifications

Re-sequencing data delivers crucial information for disease pathways analysis, breeding studies, production strain optimization or metabolic engineering.

Our re-sequencing service comprises the following steps:

• Fragmentation of the genomic DNA ligation of sequencing adaptors
• Preparation of 2 shotgun libraries (i.e. with 200 bp and 400 bp fragment size) per sample (upon request)
• Shotgun sequencing on NovaSeq, Sequel, HiSeq 2500 or NextSeq 500 or MiSeq to 30-50-fold sequence coverage
• Combination with sequencing of different Mate pair / LJD libraries (upon request)
• Mapping of the sequencing data onto a reference sequence using appropriate software (reference guided assembly)
• Genome comparison by SNP and InDels analysis
• Sequence reads are not present on the reference genome (e.g. due to phage insertions or plasmids) are automatically sorted and will be delivered

Methodology

We accept NGS grade high quality gDNA, tissue samples for this Service. We also offered DNA and RNA isolation with our validated processes for various sample sources.

Sample Requirement:

• Tissue: Minimum 3-5 gm of tissues dissect aseptically followed by fast frozen in liquid nitrogen or in RNA later shipped in dry ice to Eurofins facility.
• gDNA: Submit 1-2 µg (3-5µg if Mate pair library is requested) of RNA-free genomic DNA (NGS grade) of high molecular weight >40kb, Nanodrop A260/280 ratio >1.8; A260/230=2.0-2.2; and at a concentration of ~50 ng/µl (dissolve in low TE buffer).

Sample Quality Control:

• RNA free NGS grade gDNA isolation will be carried out using commercially available DNA isolation kit.
• The qualification and quantification of gDNA will be attained by agarose gel electrophoresis and NanoDrop/Qubit Fluorometer analysis, respectively.
• DIN value estimation will be carried out by Agilent 4200 Tape Station.

Library Preparation:

• Preparation of shotgun sequencing libraries using Illumina TruSeq Nano DNA kit.
• Long jumping distance library/MatePair library can be prepared upon request with a bridge gap of 3-5kbp.
• Prepared libraries QC will be carried out using Agilent 4200 Tape Station.

Sequencing:

• Prepared libraries will be sequenced on Illumina NextSeq500 platform with 2 x 150 bp v2 chemistry or HiSeq2500 with 2 x 125 bp v4 chemistry and NovaSeq with 2x150 bp chemistry.

Deliverables :

• Raw data will be available for download as a compressed archive of FASTQ files for each sample.
• Comprehensive compiled report and data set will be shared in link or Pen Drive or Hard Disk.
• For analysis please visit Bioinformatics Services

Turnaround time

6-8 weeks after clearance of QC process. It depends upon data size, scope, technology selected, number of samples and complexity of the project. The TAT is offered expecting no biological or technical difficulties for processing of all project samples

WGS Metagenome Sequencing

Metagenomics is a relatively new but fast growing field within environmental biology directed at obtaining knowledge on genomic information of environmental microbes as well as of entire microbial communities of particular subset of environmental condition. To identify metabolic and other functional capabilities of the microorganisms, metagenomics sequencing by shotgun sequencing strategies aims to explore all most all genes from a community and can produce detailed metabolic and functional profiles. With the sequencing technologies improving steadily, generating large amounts of sequence is becoming routine.

Eurofins Genomics India offers all comprehensive end-to-end solution for metagenomic DNA extraction from various environmental sources including soil, water, sludge, ocean sediments, rhizoosphere to shotgun library preparation and data generation using high end Illumina instruments. Our expert scientist suggest better technological tailoring depending on nature of samples.

Methodology

Sample requirement:

• Meta gDNA: Submit 2-5µg of high quality RNA-free meta-genomic DNA from soil/environmental source, NanoDrop A260/280 ratio >1.8; A260/230=2.0-2.2; and at a concentration of ~50 ng/µl.
• Soil Sample: 10-20 gm soil samples should be provided in cool pack.
• Sludge/Water: 100-200 ml of water or sludge samples shipped in cool pack to Eurofins facility.
• Plant/ Human Microbiome: 10-20 gm samples should be provided in cool pack.
• Skin Microbiome: The success of good quality of data and reads depend on the collection of high mass of pathogens. Collect skin bacteria by swabbing method by designate 4.4x4.4-cm square of skin area. Gently swab with sterile cotton swab soaked in 0.9% of sodium chloride with 0.1% Tween-20 by Z stroke manner. Transfer swab head in sterile saline solution and shipped in cool pack to Eurofins facility.

Sample Quality Control:

• High quality meta-genomic DNA will be isolated from the supplied sample using manual or commercially available DNA isolation kit.
• The qualification and quantification of gDNA will be carried out by agarose gel electrophoresis and NanoDrop/Qubit Fluorometer analysis, respectively.

Library Preparation:

• Depending on quantity of the DNA, indexed paired end meta genome library will be prepared using TruSeq Nano DNA library preparation kit or Nextera XT library preparation kit from illumina.
• Library validation will be carried out using Agilent 4200 Tape Station.

Sequencing:

• Prepared library will be sequenced using 2 x 150 bp paired end sequencing on NextSeq500 or 2 x 125 bp paired end sequencing using latest sequencing chemistry on HiSeq 2500 or NovaSeq with 2x150 bp chemistry.

Deliverable:

• Raw data will be available for download as a compressed archive of FASTQ files for each sample.
• Comprehensive compiled report and data set will be shared in link or Pen Drive or Hard Disk.
• For analysis please visit Bioinformatics Services

Turnaround time

6-8 weeks after clearance of QC process. It depends upon data size, scope, technology selected, number of samples and complexity of the project. The TAT is offered expecting no biological or technical difficulties for processing of all project samples.