GENE3340 NGS wet assessment 2017 JC v4 PDF

Preview

Summary

Download GENE3340 NGS wet assessment 2017 JC v4 PDF

Description

GENE3340 – NGS Wet Lab Assessment 2017 Name: MARIA PHAM Student #: 21513247 The aim of these questions is to assess your understanding of laboratory techniques and your ability to find information using product guides or other resources at your disposal. They may be answered as you work through the pracs, during waiting steps during wet labs, in the dry lab sessions when you have extra time, or at home. Note that the supplied resources may be sufficient to answer some of the questions, but you will likely have to draw on other sources for others or for more in-depth explanations. It is important that you develop these skills so that you are able to understand the purpose of particular steps/reactions and their components. If you carry on to do research it is likely that you will need to use product resources to help construct or troubleshoot protocols. Additionally, it is important to understand the limitations of particular kits or procedures and understand why you observe a particular outcome from experiments. Session 1: Preparation of PCR amplified rs4897612 Question 1: What is Triton X-100 and what is its purpose in the provided protocol (Section 1A)? [1 mark] Triton X-100 is a non-ionic surfactant and emulsifier which is often used in biochemical applications to solubilize proteins. Helps suppress secondary structure formation and help to stabilise the DNA polymerase. Question 2: Using the Promega BioMath calculator (online), determine the GC content and Tm for each of the primers (oligos) used in your PCR (Section 1B). [2 marks] Forward primer GC content: 41% Tm: 68 ˚C Reverse primer GC content: 31% Tm: 66˚C

Session 2: NGS Library Preparation Question 3: Write out your quantification values for your PCR products from the Nanodrop and Agarose gel. Compare these two quantification values. List at least one technical reason why either of these methods might lead to inaccurate quantification (Section 2A). [2 marks] Nanodrop: 13.7 ng/uL Agarose gel: The Nanodrop may be overstating the results, which is a common occurrence in most spectrometers. This may be a technical reason that would lead to inaccurate/imprecise quantification. Electrophoresis has a low throughput and it does not produce data very rapidly. The measurements aren’t very precise as they are subjected to many levels of human error (though out the whole process). Question 4: Why is it desirable to wait until your PCR products have been purified before quantifying (Section 2B)? [1 mark] Purification of PCR products is generally not necessary. It is advantageous to gel-purify the target DNA fragment if the PCR product is contaminated by either non-specific amplification products, primer-dimers or large quantities of unused PCR products.

Question 5: Briefly describe the three main activities occurring in the End-Repair reaction (Section 2C). [3 marks] 1. Mixing of reagents – library construction

2. 3.

a. Blunt-ended fragments are created by filling in or chewing back 3´ and 5´ overhangs. Phosphorylation of the 5´ ends ensures the fragments are suitable for ligation Incubate at 25 ˚C for 20 minutes inactivation of enzymes by incubation at 70˚C on the heating block for 10 minutes

Question 6: Why do adapters ligate only in one orientation with respect to the PCR product? Why don’t adapters ligate to each other (Section 2D)? [2 marks] Adapters ligate in one orientation to produce an orientated library so that sequencing always reads from 5’ end to 3’ end with respect to the PCR product. Adapters don’t ligate to each other because they have blunt ends.

Question 7: Describe the purpose of Bst 2.0 DNA polymerase in the barcode addition procedure. What is the purpose of the warm start (Section 2D)? [3 marks] The purpose of Bst shows improved amplification speed, yield and salt tolerance, fast polymerisation. Optimized for Loop-Mediated Isothermal Amplification (LAMP). Consistent amplification performance with the convenience of room temperature setup. WarmStart® technology eliminates off-target amplifilcation and offers increased reaction efficiency. Optimal reaction performance from 60-72 °C Session 3: Amplification and Quantification of NGS Library Question 8: Why might DNA elution be performed using a buffer with no EDTA (Section 3A)? [1 mark] EDTAmayi nhi bi tsubsequentenzy mat i cr eact i onss oi tmaybebet t ert onotus ei twhenel ut i ngt heDNA

Question 9: What is the purpose of using Q5 polymerase instead of regular Taq polymerase for amplification (Section 3B)? [1 mark] Q5 polymerase yields a significantly lower number of errors than Taq polymerase, and thus has a better proof reading ability. Using Q5 will also result in blunt ends, whereas Taq polymerase will produce single base 3’ overhangs.

Question 10: What size DNA fragments may be purified using a QIAquick PCR purification kit? Are oligonucleotides (i.e. primers) removed by this kit (Section 3C)? [1 mark] 100bp to 10kb fragments may be purified using the QIAquick PCR purification kit. It will remove primers.

Question 11: What is the dsDNA detection limit of the NanoDrop Lite? How does this compare to the Qubit dsDNA HS assay kit? Which method do you think is better for quantifying samples that need to be significantly diluted (e.g. for NGS library prep; 26 pM for IonTorrent)? Which do you think would be better for quantifying highly concentrated DNA (e.g. 1 µg/µL) (Section 3D)? [2 marks] NanoDrop Lite detection limits – 4.0 ng/uL – 1500 ng/uL Qubit dsDNA HS assay kit - 10 pg/µL to 100 ng/µL Nanodrop would be better for highly concentrated DNA. Qubit would be better for significantly diluted samples.

Resource List: Promega BioMath Calculator: http://www.promega.com/a/apps/biomath/?calc=tm GoTaq Flexi DNA Polymerase – Manual (Promega) KAPA Adapter Kit Ion Torrent – Data Sheet (Kapa Biosystems) NEBNext for Ion Torrent – Manual (New England BioLabs) Ion Plus Fragment Library Kit – User Bulletin (Life Technologies) QIAquick PCR Purification Kit – Manual (QIAGEN) NanoDrop Lite Spectrophotometer – User Guide (Thermo Fisher) Qubit dsDNA HS Assay Kit – User Guide (Thermo Fisher) Qubit dsDNA BR Assay Kit – User Guide (Thermo Fisher)...