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LIBRARY PREPARATION NGS AND NEXT GENERATION SEQUENCING

 

Next-generation sequencing technologies provides deep and precise profiling of genome and transcriptome. GENXMAP, expert in sample preparation, proposes the library preparation for sequencing purposes, but the sample preparation process varies depending on the sample type and the purpose of the experiment. Different types of genetic material (DNA or RNA) have different sample preparation processes. On top of that, the different applications of NGS add another dimension. Therefore, there are several questions that need to be asked before the experiment to determine the best methods to optimize the most the library preparation process.  The goal of library preparation is to convert the extracted nucleic acids into a format that is appropriate for the sequencing technology fits the most to the purpose of study. This is done by fragmenting the targeted sequences to a desired length, followed by adding specific adapter sequences to the ends of these targeted fragments. The adapters may also include barcodes, which identify specific samples and permit multiplexing. The fragmentation can be done by physical or enzymatic methods. No matter the type of library preparation, the purification of the library (library clean-up) and quality control step are essential before proceeding to the sequencing. 

Example of the library preparations proposed by GENXMAP and their applications:

  • Metagenomics Analysis

Metagenomics refers to the genomic analysis of microbial DNA from environmental communities using Metagenomics tools that enable the analysis of un-culturable or previously unknown microbes. For any metagenomics analysis, a reliable DNA purification step from microbial mass is essential for the analysis. Isolation and extraction must yield high quality nucleic acid for subsequent library preparation and sequencing. There are different methods for analyzing the microbial population, shotgun metagenomics or 16s sequencing.

 

  1. Shotgun metagenomics

Shotgun metagenomics is a sequencing method for environmental samples to examine thousands of organisms in parallel and sample all genes, providing insight into biodiversity and function of the selected mass. GENXMAP chooses the optimized for sample library preparation method to guaranty the amplification of low abundant targets during the library preparation, which is one of the most challenging considerations in metagenomics analysis.

Our process:

  1. Sample QC at the reception

  2. DNA Purification 

  3. DNA quantification and qualification

  4. Library preparation 

  5. Library QC

  6. Sequencing (250-300 PE) 

  7. Data Analysis/ bioinformatics analysis

  8. Reporting

 

   2. 16s Library preparation and sequencing 

Shotgun metagenomics is the study of the functional genomes of microbial communities while 16S sequencing purpose is to provide a phylogenetic analysis based on the diversity of a single ribosomal gene, 16S rRNA, that is used as the taxonomical genomics marker in bacteria and archaea. 

Our process:

  1. Sample QC at the reception

  2. DNA Purification

  3. DNA quantification and qualification

  4. Library preparation using primers for variable regions V3 and V4 of the 16S gene

  5. Library QC 

  6. Sequencing (150PE) 

  7. Data Analysis/ bioinformatics analysis

  8. Reporting

  • Whole Genome Analysis

Whole genome sequencing (WGS) refers to the comprehensive examination of a genome by reading and stitching together short (150 bp, with ILLUMINA technology) or long (up to 30kb with Oxford nanopore technology) fragments to determine an organism’s complete chromosomal (nuclear) and/ or mitochondrial DNA sequence. This technique is usually used to determine unknown genome or organism. De novo sequencing refers to sequencing a novel genome when a reference or template sequence is not available. Once a de novo genome has been completely sequenced, assembled, and annotated, a draft or common reference sequence is generated. 

Our process:

  1. Sample QC at the reception

  2. DNA Purification

  3. DNA quantification and qualification

  4. Library preparation using Illumina TruSeq DNA PCR-Free, PacBio DNA Template Prep (for the PacBio platform), Oxford Nanopore Ultra-Long DNA Sequencing Kits. 

  5. Library QC

  6. Sequencing 

  7. Data Analysis/ bioinformatics analysis

  8. Reporting

  • Whole Exome analysis 

Exome is a term used to describe the sum of all regions in the genome comprised of exons. Exome sequencing is a capture-based method developed to identify variants in the coding region of genes that affect protein function. The typical workflow required to sequence and analyze an exome is as follows:

Our process:

  1. Sample QC at the reception

  2. DNA Purification

  3. DNA quantification and qualification

  4. Library preparation using Agilent SureSelect, IDT xGEN Exome Panel, Illumina Nextera Rapid Capture Expanded Exome kits or Illumina TruSeq Exome

  5. Library QC

  6. Sequencing 2x100 or 2x150 PE

  7. Data Analysis/ bioinformatics analysis

  8. Reporting

 

  • Whole Transcriptome and mRNA library preparation and Sequencing 

The term RNA-Seq refers to a next generation sequencing approach that offers a snapshot of the entire transcriptome or messenger RNA (mRNA) profile at a given moment in time. RNA-Seq allows the detection of transcript isoforms, allele specific gene expression, gene fusions, and single nucleotide variants, all without the need for knowing anything about the sample’s sequence composition.

Most applications of RNA-Seq fall under two broad categories: 

 1. Whole transcriptome sequencing

Whole transcriptome sequencing is a snap-shot measurement of the complete transcripts in a cell, including mRNA and all non-coding RNAs. Whole transcriptome, enables to determine global expression levels of each transcript, identify exons, introns and map their boundaries. To accurately look at the whole transcriptome, most library preparation protocols first start with the removal of ribosomal RNA (rRNA) which otherwise takes up the majority of all sequencing reads. Assuming you’re not interested in ribosomal RNA, removing these transcripts allows for more of the sequencing reads to be focused on transcripts you’re actually interested in sequencing, giving you improved sensitivity toward low expressed transcripts. GENXMAP process in whole transcriptome sequencing is as below; ​

 

 2. Messenger RNA-Seq or mRNA-Seq

Messenger RNA-Seq or mRNA-Seq is a targeted RNA-Seq protocol that enriches for all polyadenylated (poly-A) transcripts of the transcriptome. mRNA-Seq is a method used in studying transcription in disease states as well as expression in variety of research based applications. Only around 1-2% of the entire transcriptome is comprised of poly-A tailed RNA, the coding part of the genome. By targeting mRNA, sequencing depth is improved as resources are dedicated to the sequencing of coding genes. This makes identifying rare variants and low expressed mRNA transcripts easier. mRNA-seq offers greater read depth than whole transcriptome sequencing.

Our process:

  1. Sample QC at the reception

  2. Total RNA Purification

  3. RNA quantification and qualification

  4. mRNA enrichment or rRNA depletion from total RNA (True Seq Stranded RNA library kits, SMARTer library preparation kits with Ribodepletion) 

  5. RNA Fragmentation

  6. Reverse transcription - 1st strand synthesis

  7. Second strand synthesis

  8. Ligation of adapters

  9. Amplification

  10. Library QC 

  11. Sequencing (100-150PE)

  12. Data Analysis/ bioinformatics analysis

  13. Reporting

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