NGS: The Basics by Sample Prep & Sequencing for ...

NGS: The Basics Sample Prep & Sequencing for Metagenomics

Stefan J. Green, PhD, Sequencing Core, Research Resources Center University of Illinois at Chicago – [email protected]

UIC SQC © ESCMID eLibrary b

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mailto:[email protected]

Disclosure of speaker’s interests

(Potential) conflict of interest

Potentially relevant company relationships in

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Fee or other (financial) payment3

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Other relationship, i.e. …5

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Please note that the use of images of specific products in this presentation does NOT represent an endorsement of that product. There are many competing instruments and reagents of high quality that are available. © ESCMID eLibrary b

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Sample Prep & Sequencing for Metagenomics

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Sample Prep & Sequencing for Metagenomics • DNA-based protocols

• Shotgun metagenome sequencing

• RNA-based protocols • Whole transcriptome sequencing


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The process…

• Define question

• Acquire samples

• Preserve samples for future, if possible

• Label samples properly; use barcoded tubes

• Extract nucleic acids and perform QC

• Prepare NGS libraries and perform QC

• Sequence and perform QC

• Data analysis

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Nucleic Acid Extraction

• Adapt protocol to tissue type, target organism, nucleic acid type, and sequencing type

• High molecular weight DNA is needed for long-read applications; enzymatic lysis

• High-energy lysis is best for microbes, but not for long-read sequencing

• Selective lysis to limit host DNA

• Automation is available


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How much DNA/RNA do I need?

• Application dependent

• Short-read technologies need much less than long-read applications

• More nucleic acid provides a wider range of possible protocols

• Realistically: • Short-read Illumina sequencing: minimum DNA – 1 ng

• Long-read Nanopore and PacBio sequencing: minimum DNA – 250 ng to 1 microgram

• For RNA, single-cell RNAseq is possible (sub-ng amounts); but poly(A) vs rRNA depletion have different requirements

• Molecular weight critical for long-read applications


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Library Preparation

• Preparing nucleic acids for sequencing is called library preparation.

• Sequencing reactions are initiated from identical regions of DNA

• Identical regions are artificially introduced using reverse transcription, PCR, ligation, or transposons.


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• DNA is fragmented into small pieces (short-read only) • Multiple options available

• Fragments are enzymatically “cleaned” up

• Sequencing adapters are ligated to fragments • Adapters are custom sequences, unique to each sequencing platform

• Adapters serve multiple purposes • Identical initiation site for primer binding

• Aid in clonal amplification

• Adapters include a sample-specific sequence known as a barcode

• Adapters MAY also include a unique molecule identifier (UMI)

• Final size selection may be performed

• Library undergoes QC and quantification

How is NGS achieved? (DNA)


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Shotgun sequencing approach for genome sequencing

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Typically, >10 kb genomic DNA fragments

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End Repair + A-tailing (sometimes)

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Shearing (acoustic or enzymatic)

Sequencing Adapter Ligation

(NGS-platform-specific)

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Shearing of gDNA


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Images of nucleic acids Genomic DNA Extracts


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Neiman et al. "Library preparation and multiplex capture for massive parallel sequencing applications made efficient

and easy." PLoS One 7.11 (2012): e48616.

T4 DNA polymerase

Taq DNA polymerase

T4 Polynucleotide kinase

T4 DNA ligase © ESCMID eLibrary b

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DNA Repair End repair is needed to prepare DNA for ligation by ensuring

that each molecule is free of overhangs, and contains 5′

phosphate and 3′ hydroxyl groups.

Step 1: Add 5 microliters of End Repair Mix to 10 microliters of sample. Mix, spin and place on ice. Step 2: Place tubes in thermocycler – 25˚C for 30 min; 70˚C for 10 min; hold at 10˚C Step 3: Spin tubes and place on ice


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DNA Ligation Step 4: Add 6 microliters of sample-specific adapter mix to each well. Step 5: Add 9 microliters of ligase mastermix to each well. Mix and spin. Step 6: – 25˚C for 30 min; 70˚C for 10 min; hold at 10˚C Step 7: Spin tubes and place on ice © ESCMID eLibrary b

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PCR Amplification Step 8: Add 70 microliters of amplification mix to each well. Step 9: Perform PCR: 5-10 cycles of PCR depending on input DNA concentration.


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Transposon-based DNA fragmentation

• Illumina Nextera • Illumina Nextera XT • Illumina Nextera FLX

“Transposases catalyze the random insertion of excised transposons into DNA targets with high efficiency.”


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• Quality analysis of samples and libraries: • Quantification of library – fluorimetry, qPCR

• Quality analysis – electrophoresis

Final Steps


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Final Steps

• Size selection • Increase distance between paired-

end reads

• Select for shorter fragments to allow paired reads to overlap

• Remove unwanted fragments

• Decrease variability in size distribution between samples

• Fragment size, or distance between adapters, is known as the insert size.


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Size Selection


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Final Steps

• Combine samples into a final ‘Pool’

• Perform quantitative PCR to measure the number of prepared molecules with adapters

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Sequencing Choices to Make

• How much data do I need? [Depth of sequencing] • Typically, 1 M to 20 M clusters of 2x150 (300 Mb to 6 Gb)

• What sequencing platform should I use?

• What read-length should I use?

• What insert size should I use?

• What kind of barcodes do I need?

• Should I use single-end or paired-end data (Illumina)?


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Sequencing Run Quality Assessment (Illumina) • Run Metrics

• Number of clusters

• Clusters passing filter

• Total yield

• % of bases with >Q30

• Error rate

• % phiX detected

• % of clusters by sample

• Caveats • Not every library type is the same

• Metrics expected for one type of library may not be achievable for others

• PF >80%

• %Q30 >75%


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Sequencing Run Quality Assessment (Illumina)


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https://blog.horizondiscovery.com/diagnostics/the-5-ngs-qc-metrics-you-should-know


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https://blog.horizondiscovery.com/diagnostics/the-5-ngs-qc-metrics-you-should-know


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• Many different protocols • Poly(A) Capture – eukaryotic organisms only • Ribosomal RNA removal – custom and pre-designed • Small RNA (e.g., miRNA) protocols

• More challenging than DNA-based protocols • Most protocols require conversion to ds-cDNA • RNA is more readily degraded • Microorganisms do not polyadenylate their mRNAs • Microorganisms do rapidly change their expression profiles • RNAseq can be confounded by residual gDNA • Ribosomes are the dominant RNA species • Microbial mRNA-seq in the presence of host RNA is tricky

How is NGS achieved? (RNA)


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RNA Quality Analysis


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RNA Quality Analysis

• RNA quality determines which RNAseq protocol can be used

• For microorganisms, poly(A) capture can never be used

• Thus, ribosomal RNA depletion must be performed (or excess sequence data generated)

https://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-22/CB22.html


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Ribosomal RNA Depletion • Multiple techniques for removal

of ribosomes • Hybridization of DNA probes,

followed by RNAse H (shown right)

• Hybridization of biotinylated DNA probes, followed by streptavidin capture

• Hybridization of probes, followed by selective restriction digest

• Double depletion needed for samples with host RNA

www.neb.com


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http://www.neb.com/

Sample mRNA workflow • RNA may or may not be

fragmented

• Reverse transcription with random primers – incorporate artificial sequence at 5’ end

• Molecular tricks to incorporate artificial sequence at 3’ end

• PCR amplify to incorporate sequencing adapters and barcode


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Microbial RNAseq

• Depth of sequencing needed • Generally, 5-10 M clusters for

single organism after ribosomal RNA depletion

• 50 M clusters or more may be needed for complex microbial communities

• Paired-end sequencing not absolutely necessary due to low intronic content

Ofek-Lalzar, Maya, et al. "Niche and host-associated functional signatures of the root surface microbiome." Nature communications 5 (2014): 4950. © ESCMID eLibrary b

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