Paths to NGS | Agilent

Immuno precipitated DNA

DNA-binding proteins (such as transcription factors and histones) can be crosslinked to the DNA they are binding within the cell. Using antibodies specific to the binding proteins, the protein-DNA complex can be immunoprecipitated/purified out of the cellular lysate, and the DNA can be separated from the protein. This immuno precipitated DNA can be used as the input for NGS. QC of this input material ensures that the sample is of good enough quality to prepare an NGS library.

Are you ready for successful library prep?

Start here

FFPE DNA

Formalin-fixed paraffin-embedded (FFPE) refers to a specific way to preserve and prepare specimens for future use, preserving the proteins and vital tissue structure. FFPE DNA is the DNA that has been recovered from FFPE tissues. Often highly degraded, FFPE DNA contains fragments that are smaller than the optimal size recommended for sequencing, and thus requires QC before library preparation to confirm that at least a portion of the sample is above 10,000 bp. Based on the amount of sample that exceeds this threshold, adjustments can be made to the sample shearing, input concentration, or library preparation protocol to help ensure the creation of a successful sequencing library.

Are you ready for successful library prep?

Start here

Genomic DNA

Genomic DNA is a nucleic acid structure that is comprised of all of an organism's biologically inherited information. Generally long in sequence, gDNA must be sheared to smaller fragment sizes to be used for short-read NGS sequencing. For library prep kits using tagmentation, accurate quantification is required to enable appropriate fragmentation. QC is essential to ensuring the sample is of good quality, size, and concentration.

Are you ready for successful library prep?

Start here

Total RNA

RNA is a single stranded nucleic acid that carries instructions from DNA in form of amino acids to the ribosomes for protein synthesis. Total RNA is all of the RNA types contained in a cell, including mRNA, rRNA, tRNA, small RNA, etc. Methods of RNA extraction can be prone to RNA degradation, making QC of total RNA crucial before beginning library preparation to ensure that you have intact, quality, highly concentrated sample.

Are you ready for successful library prep?

Start here

FFPE RNA

Formalin-fixed paraffin-embedded (FFPE) refers to a specific way to preserve and prepare specimens for future use, preserving the proteins and vital tissue structure. FFPE RNA is the RNA that has been recovered from FFPE tissues. Because they are highly degraded and contain fragments that are smaller than the optimal size range for sequencing, FFPE RNA samples require QC before use in library preparation protocols. QC helps to ensure the sample meets a defined quality - samples with at least 30% of the fragments being above 200 nucleotides are suitable for sequencing - and lets you know if any modifications need to be made to the sample shearing, input concentration, or library preparation protocol. By making necessary adjustments, high-quality sequencing results can still be achieved from low-quality FFPE samples.

Are you ready for successful library prep?

Start here

mRNA

Messenger RNA (mRNA), is a type of RNA that transports the genetic information from DNA to the ribosomes for protein synthesis. mRNA sequencing provides information about the transcriptome and gene expression. As mRNA is enriched from total RNA, QC of the mRNA is important to ensure that your mRNA is of good quality and sufficient concentration for library preparation, and to identify any residual rRNA contamination. The integrity of mRNA samples is crucial to obtaining meaningful sequencing results, as sequencing degraded samples may exclude certain transcripts and result in misinterpretation of gene expression patterns.

Are you ready for successful library prep?

Start here

Ribo-depleted RNA

Ribo-depleted RNA is RNA that has been enriched for whole transcriptome RNA by depleting the ribosomal RNA (rRNA) species present. rRNA is the most abundant type of RNA, and thus is often overly represented in RNA-sequencing studies, necessitating ribo-depletion of RNA before sequencing for proper representation of the transcriptome. As an input into RNA-sequencing, ribo-depleted RNA must undergo QC in order to confirm sample integrity, identify any residual rRNA contamination, and determine sample concentration before beginning the library preparation process.

Are you ready for successful library prep?

Start here

R.E Digest

A restriction enzyme (R.E.) is an endonuclease that works by cleaving DNA at a specific sequence. R.E. digestion in NGS library preparation is a way to fragment gDNA to a desired size, with sticky or blunt ends that enable the DNA fragment to ligate to an adaptor.

Are you ready for successful library prep?

Start here

Ligate Adaptors

Adaptors are short, double-stranded pieces of synthetic DNA. DNA ligase is an enzyme that joins DNA strands together. During the process of adaptor ligation, these oligonucleotide adaptors, specific to the sequencing platform, are ligated to the fragmented sample. QC at this step ensures that the adaptor has been successfully ligated to the DNA, and that enough adaptor-ligated DNA still remains following clean-up to proceed with library preparation.

Are you ready for successful library prep?

Start here

Fragmentation

Fragmentation is the process of shearing the input sample into smaller pieces, or fragments. This can be done by several methods, including enzymatic or mechanical (sonication, needle-shear, etc.). Samples should be fragmented to the desired insert size (the portion of the library between the adaptors), and QC is an important step here to ensure that the sample is sized correctly, not too small or too long. For transposase-based methods of fragmentation, this step also includes adaptor ligation.

Are you ready for successful library prep?

Start here

Post insertion clean-up

This step is to purify the DNA that has undergone enzymatic fragmentation in order to eliminate any remaining enzymes that could interfere with downstream steps. QC after any clean-up step is highly recommended to ensure that the sample was not lost during clean-up, and that the clean-up was successful.

Are you ready for successful library prep?

Start here

Amplify to index

An index, or barcode, is a specific sequence that can be given to each library to enable multiplexing of several libraries for one sequencing run. Barcodes ensure that all fragments from each individual library have the same index and can be separated during data analysis. Amplification, or making multiple copies, of the indexed samples is required so that the sequencer has a strong enough signal to detect each library. QC at this step will show a slight size shift compared to the adaptor-ligated sample and enable quantification of the library, ensuring that the library has been amplified enough for sequencing.

Are you ready for successful library prep?

Start here

Hybridize and capture

This step is specific to capture-based sequencing protocols to enrich targeted gDNA. Biotinylated probes created to target specific regions of the gDNA hybridize to the sample. The probes, along with the targeted regions, are then pulled down via magnetic beads, resulting in only libraries enriched for the target region.

Are you ready for successful library prep?

Start here

Uracil digest

During this process, the uracils that were added to the second strand cDNA are enzymatically removed in order to facilitate PCR enrichment.

Are you ready for successful library prep?

Start here

Exonuclease digest

An exonuclease digest eliminates excess probes or oligos from the sample, leaving behind only the adaptor ligated DNA for further PCR amplification.

Are you ready for successful library prep?

Start here

Template purification

A clean-up step to purify each individual library before pooling.

Are you ready for successful library prep?

Start here

End repair

During the process of fragmentation, the resulting double-stranded fragments do not always completely align on the ends. Thus, after fragmentation, the DNA is converted to blunt ends using specific enzymes to remove 3' overhangs or fill in 5' overhangs.

Are you ready for successful library prep?

Start here

Adenylation

Adenylation is the addition of a deoxyadenosine 5'-monophosphate (dAMP) to the 3' ends of the fragmented samples, forming a poly(A)-tail that aids in adaptor ligation.

Are you ready for successful library prep?

Start here

Adaptor ligation

Adaptors are short, double-stranded pieces of synthetic DNA. DNA ligase is an enzyme that joins DNA strands together. During the process of adaptor ligation, these oligonucleotide adaptors, specific to the sequencing platform, are ligated to the fragmented sample. QC at this step ensures that the adaptor has been successfully ligated to the DNA, and that enough adaptor-ligated DNA still remains following clean-up to proceed with library preparation.

Are you ready for successful library prep?

Start here

Enrich

PCR amplification to generate more copies of only the sequences that contain adaptors on both ends. QC at this step includes a visual inspection of the library to ensure correct sizing and indicate if a clean-up step is required to eliminate any primer dimer or PCR artifacts. The presence of these artifacts may indicate that the PCR conditions are not optimized and give researchers an indication that further method validation may be necessary for future preparations. Additionally, quantification of the library at this point is important to enable normalization of individual libraries for multiplexing.

Are you ready for successful library prep?

Start here

Normalize / Pool

At the end of library preparation, the sample must be quantified to determine the amount to load onto the sequencer. In the event that multiple samples will be sequenced together, the samples can be "normalized," or diluted to the same molarity, and mixed together. This final pooled library must undergo QC to ensure the sizing distribution and molarity before sequencing.

Are you ready for successful library prep?

Start here

To NGS platform

The input DNA has now been ligated to its adapters, PCR amplified, normalized, and the libraries are nearly ready to go! The QC checkpoints built into your workflow have ensured you can be confident in your libraries. Your libraries are now ready for sequencing, either manually or via an automated NGS prep system.

What solution works best for you?

Take the next step and find out.

1st Strand cDNA

cDNA is a complementary DNA strand. To sequence RNA, it must first be converted to cDNA by reverse transcription so that the sequence can be PCR amplified. cDNA is generated by pairing RNA base pairs to their DNA complements.

Are you ready for successful library prep?

Start here

2nd strand cDNA

2nd strand cDNA synthesis is the process of generating double-stranded cDNA from the first strand cDNA template. During this step, the RNA is completely removed and replaced with DNA to enable sequencing.

Are you ready for successful library prep?

Start here

2nd strand cDNA w dUTP

The purpose of this step is to selectively remove one of the two cDNA strands, by incorporating dUTP into the second strand cDNA synthesis reaction, for strand-specific library construction.

Are you ready for successful library prep?

Start here

Target Enrichment

Regions of gDNA can be selectively targeted by using sequence-specific primers and probes that anneal to the target amplicon. These targeted sections of gDNA can then be amplified by PCR and sequenced, ensuring that the final library consists only of the targeted sequences.

Are you ready for successful library prep?

Start here

Hybridize and capture

This step is specific to capture-based sequencing protocols to enrich targeted gDNA. Biotinylated probes created to target specific regions of the gDNA hybridize to the sample. The probes along with the targeted regions are then pulled down via magnetic beads, resulting in only libraries enriched for the target region.

Are you ready for successful library prep?

Start here

Size selection

Size selection is the process of sizing the target sequences to a desired length. During this step, the size is refined and artifacts such as adaptor and primer dimers are removed from the library. QC is critical at this step to ensure that a library was successfully made, is of the correct size and sufficient concentration for sequencing, and that artifacts that may inhibit sequencing have been removed.

Are you ready for successful library prep?

Start here

Amplify to index

An index, or barcode, is a specific sequence that can be given to each library to enable multiplexing of several libraries for one sequencing run. Barcodes ensure that all fragments from each individual library have the same index and can be separated during data analysis. Amplification, or making multiple copies, of the indexed samples is required so that the sequencer has a strong enough signal to detect each library. QC at this step will show a slight size shift compared to the adaptor-ligated sample and enable quantification of the library, ensuring that the library has been amplified enough for sequencing.

Are you ready for successful library prep?

Start here

Strand displacement

Enzymatic fragmentation can leave behind a small single-stranded gap in the dsDNA. Strand displacement is the process of repairing this gap to ensure that the DNA can be prepared for circularization.

Are you ready for successful library prep?

Start here

Circularization

Following strand displacement clean-up, this step ligates the DNA fragments into circular molecules.

Are you ready for successful library prep?

Start here

Digest linear DNA

This step uses an exonuclease to remove any leftover linear DNA from circularization steps, leaving only circularized DNA.

Are you ready for successful library prep?

Start here

Shear circular DNA

After digesting linearized DNA, the remaining circularized DNA is digested into smaller fragments, generating 3' and 5' overhangs.

Are you ready for successful library prep?

Start here

Bind DNA

After shearing of circularized DNA, streptavidin beads are added to purify only the DNA fragments that contain adaptors, allowing for removal of fragments without adapters.

Are you ready for successful library prep?

Start here

Featured

What Path Are You On?

Interactive Map

Paths to NGS Poster