Review Questions
Review Question 1
Why should a final library have a median insert size of ~250–300 bp to support long
paired end 2 Â 150 read lengths?
Review Question 2
Which different read-outs can be obtained by different RNA-Seq Library Preparation
methods?
Review Question 3
A crucial factor leading to misrepresentation of data is the bias prevalent in almost all
steps of NGS sample preparation. Discuss a few possible solutions to this kind of bias!
Review Question 4
Graphically illustrate the structure of adapter-lying DNA/cDNA fragments and label
the individual sequence sections of the adapters!
Answers to Review Questions
Answer to Question 1: Otherwise the percentage of adapter contaminated reads
increases;
Answer to Question 2:
Objective
Principles of approach
Gene expression
Target poly(A) mRNAs (enrich or selectively amplify)
Alternative
splicing
Target exon/intron boundaries by either long-read sequencing (>300 bp)
or paired end sequencing (!2 Â 75) and rRNA depletion
miRNA (or small
RNAs)
Target short reads (miRNAs: 18–23 bp) using size selection purification.
piRNAs, snoRNAs, tRNAs are all <100 bps
Answer to Question 3: Bias during amplification of AT- and GC-rich regions: PCRfree amplification could yield better read distribution and coverage compared to PCR
methods, but would require large quantities of starting DNA material.
PCR bias during library preparation for RNA-Seq can be introduced by the additional
steps to convert RNA to cDNA. KAPA HiFi DNA polymerase can be used for the
amplification step to reduce this kind of bias.
44
M. Kappelmann-Fenzl
Review Question 1
Why should a final library have a median insert size of ~250–300 bp to support long
paired end 2 Â 150 read lengths?
Review Question 2
Which different read-outs can be obtained by different RNA-Seq Library Preparation
methods?
Review Question 3
A crucial factor leading to misrepresentation of data is the bias prevalent in almost all
steps of NGS sample preparation. Discuss a few possible solutions to this kind of bias!
Review Question 4
Graphically illustrate the structure of adapter-lying DNA/cDNA fragments and label
the individual sequence sections of the adapters!
Answers to Review Questions
Answer to Question 1: Otherwise the percentage of adapter contaminated reads
increases;
Answer to Question 2:
Objective
Principles of approach
Gene expression
Target poly(A) mRNAs (enrich or selectively amplify)
Alternative
splicing
Target exon/intron boundaries by either long-read sequencing (>300 bp)
or paired end sequencing (!2 Â 75) and rRNA depletion
miRNA (or small
RNAs)
Target short reads (miRNAs: 18–23 bp) using size selection purification.
piRNAs, snoRNAs, tRNAs are all <100 bps
Answer to Question 3: Bias during amplification of AT- and GC-rich regions: PCRfree amplification could yield better read distribution and coverage compared to PCR
methods, but would require large quantities of starting DNA material.
PCR bias during library preparation for RNA-Seq can be introduced by the additional
steps to convert RNA to cDNA. KAPA HiFi DNA polymerase can be used for the
amplification step to reduce this kind of bias.
44
M. Kappelmann-Fenzl