Sequencing and Bioinformatics

Question 1

Read length

Answer 1

of bases sequenced for a DNA fragment (provided as a maximum or mean)

Question 2

Read depth

Answer 2

of times a nt is read during sequencing. A high read depth reduces errors

Question 3

Reads per run

Answer 3

of sequences produced per run (usually provided as a maximum)

Question 4

Accuracy (sequencing)

Answer 4

% error rate of an instrument (usually provided as 100% - error rate);
error includes substitition, base-specific bias, etc.

Question 5

Time per run

Answer 5

Average time for a run in an HTS (High-Throughput Sequencing) instrument

Question 6

Cost per million bases

Answer 6

Very variant across time and geographic region

Question 7

Short-read instruments

Answer 7

300-500 bp reads (e.g. Illumina and Ion sequencing)

Question 8

Long-read instruments

Answer 8

>50kbp (e.g. SMRT, Nanopore)

Question 9

Pyrosequencing (Microfabricated picolitre reactors)

Answer 9

Emulsion PCR in droplets inside beads;
Known NT’s are flowed and washed away;
Optic slide sensor camptures emitted photons when a nt is incorporated;
1 M reads occur sumultaneously (i.e., parallel sequencing);
Sequences are put together as contigs (de novo);
96% - 98% accuracy

Question 10

ion torrent (non-optical semiconductor-device)

Answer 10

Amplification occurs on beads inside wells;
Nucleotides are flowed step-wise;
Chip detects H ions released by DNApol upon incirporating nt’s as pH shift;
Reads occur simultaneously (i.e. parallel sequencing):
99.9% accuracy

Question 11

Illumina sequencing

Answer 11

barcodes are placed on adaptors;
libraries prepared in 94 wells;
flow cell (glass slide) with lanes containing bound oligos;
bridge amplification (forward and reverse) repeated many times;
each nt has acgaracteristic fluorescence signal (sequencing through synthesis);
base call determined by wavelenght emission and signal intesnity;

Question 12

Single-Molecule Real-Time (SMRT)

Answer 12

a single molecule is immobilized in a nanophotonic structure;
wavelenght is detected by feluorophore laser excitation;
each dNTP has a different fluorophore and emits a didferent color;
very fast and cheap

Question 13

Single-molecule nanopore DNA sequencing (Nanopore)

Answer 13

Does not require sample amplification;
Does not require fluorescent labelling;
ssDNA molecule passes through a protein nanopore;
an adaptor on the membrane detects ionic current passing through pore;
each nt has a different ionic current;

Question 14

Sagner sequencing (Manual Sanger dideoxy chain terminator DNA sequencing)

Answer 14

reactions happen inside microcapillaries;
fluorecent ddNTP (H instead of OH on 3rd C of ribose) makes amplification stop;
Laser excites fluorescin on each ddNTP;
Each ddNTP has a different wavelenght;
wavelength indicates at which nt amplification stopped;
output is electropherogram;

Question 15

RESCRIPt

Answer 15

open source language that compiles into javascript

Question 16

QIIME2

Answer 16

“quantitative insight into microbial ecology”
Open source, commnity-developed bioinformatics pipeline

Question 17

BOLD

Answer 17

Barcode of life datasystem;
A dataset of DNA barcode records that also ha morphological, geographic, and taxonomic data for species

Question 18

sequence filtering

Answer 18

selection of sequences by desired characteristics like unambinguity, homopolymers, and length

Question 19

sequence dereplication

Answer 19

deletion of duplicated sequences

Question 20

naive Bayes classifier object

Answer 20

A probabilitic algorithm used for classifying and clustering; it’s call naive becuase it’s based on the assumption of independence, which we don’t care about in this case

Question 21

Cytochrome Oxidase I

Answer 21

A mitochodnrial DNA sequence commonly used as a barcode; in nature it codes for a protein used in respiration

Question 22

16S rRNA

Answer 22

encodes the ribosomal subunit, used in translation

Question 23

bold R library

Answer 23

R package used to access sequences rom BOLD

Question 24

gaps (sequencing)

Answer 24

nucelotides that have been removed from a sequence

Question 25

.fasta

Answer 25

file extension for a text file contining a genetic or aminoacid sequence

Question 26

metadata

Answer 26

file that has informaiton accompanying sequences like place collected and taxonomy info

Question 27

.csv

Answer 27

file extension of a table converted to text; stands for “comma separated values”

Question 28

IUPAC characters

Answer 28

16-character code that allows the specification of ambigupus nucleic acids

Question 29

.qza

Answer 29

file extension used by QIIME

Question 30

homopolymer

Answer 30

a sequence of repeated nucelotides

Question 31

leading/trailing

Answer 31

first/last nucleotides in a sequence

Question 32

ambiguous nucleotides

Answer 32

nucelotide entry in a sequence that is neither ATCG

Question 33

alignment

Answer 33

Process to arrange sequences in order to identify regions of similarity

Question 34

upstream/downstream

Answer 34

upstream: towards 5’
downstream: towards 3’

Question 35

MAFFT

Answer 35

a program for performing multiple sequence alignment

Question 36

sequence trimming

Answer 36

removing bits of sequence before and after a region of interest