midterm 2 lecture questions Flashcards
In Illumina NGS (sequencing by synthesis) what is the ratio of
dNTP:ddNTP is added to the flow cell during each cycle? Explain why:
100:0
90:10
50:50
10:90
0:100
Rationale:
0:100 because dNTPs will not emit any fluorescence and will continue being polymerised instead of halting the chain
You are conducting a study on bacterial diversity in the soil around different
types of trees on campus. You collect 15 samples from each type of tree and
use amplicon sequencing to identify count species of bacteria at the different
sites. Similar numbers of reads are obtained per sample. You generate
rarefaction curves (one with plateau, one without), What are some interpretations you might make about the sampling protocol for the different species of tree? What are some things this is suggesting?
no plateau = sampling is not completed and full richness is not displayed, don’t know which is truly more diverse until both plateau
Bonus: why is it important that the question states that similar number of
reads were obtained for each sample? How would things change if there were differences?
if not stated, diversity differences could’ve been due to sequencing errors, detecting fewer species than expected
DADA2 is part of what type of workflow?
-Whole genome sequencing
-Shot-gun metagenomic sequencing
-Amplicon sequencing
-Meta-transcriptomic sequencing
-amplicon sequencing
(T/F) The NEJM paper we looked at demonstrated that higher levels of beta
diversity at initial admission or during engraftment are associated with better
outcomes during hematopoietic stem cell transplant.
false - looked at alpha diversity
When performing meta-transcriptomic or transcriptomic analysis, one of the
common steps is to deplete rRNA from your RNA sample. Why do you think
that might be the case?
80% of RNA is rRNA, and will encode the same things so amplifying it will just deplete resources
Your colleague shows you the following graphs, and says that they want to
study how Actinobacteria use antibiotics to fuel their growth and expansion.
What is your response to them? What might you tell them to do (and how)?
-cannot make any comparisons with relative expression, need sample unit and rarefaction curves to ensure the sampling covered all species richness
(T/F) Since they all belong to the same species, different isolates/strains ofE. coli use of the same sugars in the gut.
false, alpha diversity represents differences within a strain
