Welcome back to abm’s Next Generation Sequencing video series, where we discuss major concepts

in NGS, such as the science behind it and how to work with sequencing data.

For all your NGS experimental needs, Applied Biological Materials, or abm, offers a wide

range of affordable services, including whole genome sequencing, RNA sequencing, exome sequencing,

and even lane rentals. In our previous video, we introduced you to

the concept of NGS, plus the methods and machines available. Here, we will outline the steps

needed to prepare samples for sequencing and the specifics for each sequencing application.

We invite you to watch our previous video before starting this one.

Before a sample can be sequenced, it must be prepared into a sample library from genomic

DNA or total RNA. A library is a collection of randomly sized DNA fragments representing

the sample input. However, depending on the type of NGS applications, different library

preparation steps are taken. There are four types of NGS applications which are considered

below: Whole Genome Sequencing (WGS), Exome Sequencing (Exome-Seq), RNA Sequencing (RNA-Seq),

and Methylation Sequencing (Methyl-Seq). Whole Genome Sequencing, or WGS, refers to

the sequencing of an organism’s entire genome. There are four different library preparation

kits for whole genome sequencing: 1) TruSeq PCR-free Library Preparation Kit:

ideal for any genome where there is 1-2 μg of genomic DNA available. The purpose of this

kit is to avoid PCR amplification errors over long distances. Genomic DNA is isolated from

the sample and fragmented physically or chemically, often creating overhangs. The resulting DNA

fragments are then purified using magnetic beads for the desired size of 350 or 550bp.

This occurs by incubating specific ratios of magnetic beads with fragmented DNA. Then,

the end overhangs are repaired into blunt ends. The 3’ ends of the fragments are additionally

adenylated with a single base, allowing hybridization to the 3’ thymine overhang of sequencing

adapters. The fragments are then ligated to the adapters. This ligation step is critical

for sequencing, as the adapters will enable the DNA to hybridize to the surface of the

sequencing reaction chip. The collection of adapter-ligated fragments forms a library,

which must be validated before being sequenced. Quantitative validation is done with qPCR,

for two reasons: 1) the adapter sequences already contain the primers used in qPCR,

allowing quantification only of the adapter-ligated fragments, and 2) the library is too small

to be quantified flurometricly, as there was no PCR amplification during preparation. Qualitative

validation is done with the Agilent Technologies 2100 Bioanalyzer, before optional pooling

with other libraries. 2) TruSeq Nano DNA Library Prep Kit: ideal

if only 100-200 ng of genomic DNA are available. The protocol is almost identical to the TruSeq

PCR-free Library Preparation Kit, except for PCR amplification and library validation.

Amplification occurs between adapter ligation and library validation steps. This is done

to enrich for adapter-ligated DNA fragments and increase the concentration of the library

for sequencing. Library validation is nearly the same as for the TruSeq PCR-free Library

Preparation Kit. However the high library concentration and, more importantly, selective

amplification of fragments ligated with correctly oriented adapters allow for fluorometric quantification.

3) Nextera DNA Library Prep Kit: ideal for large, complex genomes, for example, the human

genome, and provides a shorter sample preparation time relative to the TruSeq PCR-free and Nano

Library Prep Kits. The protocol is similar to the TruSeq Nano DNA Library Prep Kit, although

with a few differences. Unlike the TruSeq kits, fragmentation and adapter ligation of

genomic DNA, also known as tagmentation, occur in the first step. This is done with an enzyme

called a transposome, which is a transposase-transposon complex. The enzyme is able to make cuts in

DNA like a transposase, but also insert a portion of itself in the DNA sequence like

a transposon. The Nextera transposome is unique as the transposon portion consists of adapter

sequences. During tagmentation, the Nextera transposome simultaneously cleaves the DNA

molecule and inserts these adapter sequences. There is a clean-up step to remove any remaining

transposome bound to the DNA from interfering with later steps. Because DNA fragmentation

and tagging occur at the same time, there is no need for DNA fragment end repair or

adapter ligation preparation. Library quantification is solely done with Qubit, while qualitative

validation is done with the Bioanalyzer. 4) Nextera DNA XT Library Prep Kit: ideal

for small genomes, for example, bacteria, as well as plasmids and amplicons. The protocol

is similar to the Nextera DNA Library Prep Kit, with two exceptions: There is neither

post-tagmentation clean-up nor library quantification. Exome Sequencing, or Exome-Seq, is the sequencing

of the coding portion of the genome. Currently, this is a more affordable alternative to whole

genome sequencing, since exome-seq only requires about 2% of the whole genome to be sequenced.

Exome-Seq can be performed in two ways: 1) Sequencing of only the exons, or 2) sequencing

of all the exons, introns, which are non-protein coding regions, and regulatory regions, such

as the 5’ and 3’ untranslated regions (5’ and 3’-UTR) and microRNAs (miRNA)

sequences. 1) Nextera Rapid Capture Exome Kit: ideal

if only the exons are to be analyzed. Like the Nextera DNA Library Prep Kit protocol

for whole genome sequencing, the first step is tagmentation of genomic DNA. This is followed

by a clean-up step where the transposome is removed to prevent interference in later steps.

In the first of three PCR enrichment step, adapter-ligated fragments are amplified to

enrich for adapter-ligated DNA, and to increase library concentration. Sequencing and indexing

priming sites are also added. Once amplification is complete, the library is purified from

non-amplified fragments with magnetic beads. The library is also quantified fluorometricly

to determine if there are enough products. Next is the isolation of exome-amplified fragments.

This is achieved by hybridizing the exome-amplified fragments to biotinylated oligonucleotide

probes, which are complementary to the exome. Non-covalent binding of biotinylated sequences

with streptavidin beads allows capture, or selection, of the fragments. During these

steps, non-specifically bound DNA is removed with washes. The process of hybridization

and capture is repeated a second time. Once this is complete, the DNA library is enriched

twice. The library is then purified with magnetic beads for a final round of enrichment, after

which the library is purified again. Finally, quantitative validation is done using either

qPCR or Qubit, while qualitative validation is performed with the Bioanalyzer.

2) Nextera Rapid Capture Expanded Exome Kit: ideal if a more complete analysis of the exome,

including UTRs and miRNA binding regions, is desired. The protocol is almost identical

to the Nextera Rapid Capture Exome Kit, except for the addition of specific probes and related

beads which bind and capture non-protein coding regions.

RNA Sequencing, or RNA-Seq, consists of sequencing the RNA transcripts present in the sample

of an organism. The sample preparation required for RNA samples vary depending on the type

of RNA. 1) TruSeq Stranded Total RNA Kit: ideal if

analyzing the transcriptome is desired. Ribosomal RNA (rRNA) is not desired in the total RNA

sample library, so it must be depleted by binding them to magnetic beads with sequences

complementary to rRNA. After hybridization, the magnetic beads are removed from the solution

and the supernatant is used in further preparation steps. The remaining RNA is cleaned, fragmented,

and primed in a single step. The first cDNA strand is then synthesized, and the compound

Actinomycin is added to prevent second strand synthesis while the first strand is made.

The RNA template is then degraded to ensure that only the second cDNA strand will be produced

in the next synthesis step. During the second cDNA strand synthesis, dUTP nucleotides are

used instead of dTTP nucleotides. The purpose is to differentiate between the two strands

of DNA after synthesis. The resulting double-stranded DNA is prepared for adapter ligation through

adenylation of the 3’ end. This allows the cDNA to hybridize with the thymine on the

3’ end of the adapters. Once adenylation is complete, adapters are ligated onto the

3’ends of the cDNA and dUTPs are enzymatically removed. Now lacking dUTPs, the fragments

are enriched via PCR amplification. The resulting library is validated quantitatively with qPCR,

and qualitatively with the Bioanalyzer before normalization. If necessary, the library can

be pooled with others for multiplexing. 2) TruSeq Stranded mRNA Kit: ideal if the

gene expression profile of a sample is desired. The protocol is identical to the TruSeq Stranded

Total RNA kit, with the exception of mRNA enrichment instead of rRNA depletion in the

first step. 3) TruSeq Small RNA Kit: ideal if small, non-coding

RNAs, for example, miRNA, are to be analyzed. The protocol for this kit is very different

from the previous RNA kits, due to the omission of RNA depletion and enrichment. Also, the

first step consists of sequential blunt-ended adapter ligation (3’ adapter then 5’ adapter)

to total RNA. The adapter-ligated RNAs are then subject to RT-PCR to enrich for RNAs

with adapters in the correct orientation. Products of RT-PCR are run on an agarose gel,

and the desired products are isolated at 147 and 157bp. The purified library is only validated

qualitatively, using the Bioanalyzer. TruSeq DNA Methylation Kit, ideal if genome

methylation is to be analyzed. The sample library preparation begins with genome fragmentation.

Once complete, the fragments are treated with bisulfite to convert non-methylated cytosines

into uracils, while retaining methylated cytosines. Using random primers containing the 5’ adaptor

sequence at their 5’ end, DNA amplification occurs. Next, the 3’ adapter tag is ligated.

PCR enrichment for the adapter-ligated fragments is performed, and if desired, indexing primers

for sequencing are added. The enriched library is purified using magnetic beads, before quantitative

validation of the library with qPCR or a fluorometric method. Qualitative validation is also performed

with a Bioanalyzer. All of abm’s NGS services include sample preparation, see a full list

of our services here. For detailed information on all the sample preparation procedures,

including flow charts and links to appropriate protocols, please view our knowledge base

here. Please leave your questions and comments below

and we will answer them as soon as possible. Thank you for watching!