-
List the four different methods for assembling clones:
- Restriction Fingerprinting
- Repetitive DNA fingerprinting
- Repetitive DNA PCR
- STS content mapping
-
Describe Maxam-Gilbert sequencing method
- Chemical degradation method
- Uses toxic chemicals
- had low throughput
- No longer in use, Sanger sequencing took over
-
What does the Phred score deliver?
The quality reading and confidence of nucleotide accuracy.
-
What sequencing technology was the first to do sequencing by synthesis?
Sanger sequencing
-
Describe how sanger sequencing does sequencing by synthesis:
- It utilizes DNA polymerase for in vitro DNA replication
- ddNTP (dideoxynucleotides) are fluorescent labeled to clearly color indicate which nucleotide they represent
- The ddNTPs lack the 3' OH groups needed for continuing the sequence, so when the fluorescence is added, the chain stops replication.
- This is then fed through capillary electophoresis to excite the fluorescence with a laser to read the sequence
-
Describe 454 pyrosequencing:
- sequencing by synthesis
- Attach DNA fragments to beads, use emulsion PCR to amplify and place beads in wells on a slide, one bead per well
- the slide is flooded with one of four known NTPs, which causes a chemiluminenscent reaction when incorporated onto the chain.
- The NTPs are washed away and a new set of NTPs are added
- The light signal can be quantified to identify if multiple of the same nucleotide were added
- has trouble differentiating the signal when (>3 are added at a time)
-
How does Illumina amplify DNA for sequencing?
Bridge amplification
-
Which company made most of the capillary machines that were used for genome sequencing in the early 2000s?
Applied Biosystems. Inventors of Solid sequencing
-
Sequencing method that measures changes in pH
Ion Torrent
-
Which sequencing method does sequencing by ligation?
SOLiD
-
Which system uses a ZMW (zero- mode wave guide)?
PacBio SMRT
-
How does oxford Nanopore sequence?
by measuring current during strand synthesis as DNA or RNA passes through a pore on the late.
-
What sequencing technology did we discuss that can detect methylation?
Oxford Nanopore
-
Which system does the minIon go with?
Oxford nanopore single molecule sequencing
-
Fastq files
stores a biological sequence and corresponding quality scores
-
Fasta files
- text-based format for representing nucleotide sequences or peptide sequences where nucleotide or amino acids are represented using single letter codes
- often a consensus sequence
-
What are Phred quality scores?
- Give a measurement of the error rate.
- Applied to the confidence that a nucleotide was correctly identified.
-
What does Ultima genomics claim their technology does?
- Remove some of the limitations of Illumina:
- Higher throughput
- cheaper
-
What type of amplification does MGI technology and AVITI (Element Biosciences) utilize?
Rolling circle amplification.
-
What are the steps of genomic sequencing?
- Library making (large insert library from a genome)
- Production sequencing (generate fragments, preform sequencing reactions, determine sequence)
- Finishing (assemble into a continuous sequence, fill gaps)
-
Which program assists in assembling sequences?
PHRAP
-
Steps of sequence analysis:
-
Sequencing alignment method: graphical approach
-
Example of dynamic programming algorithms for sequence alignment (2)
- Needleman-Wunsch (global alignments)
- Smith-Waterman (local alignments)
-
Example of Heuristic approach of sequence alignment
-
Describe a hidden Markov approach to annotating the genome
- A hidden Markov model (HMM) is a statistical model that can be used to describe the evolution of observable events that depend on internal factors, which are not directly observable
-
Provide an example of a gene prediction routine:
- 1. Training of Hidden Markov Models
- 2. Employment of dynamic programming to score all possible gene models
- 3. Usage of EST information to retrain models and refine gene predictions
-
What are ESTs?
- Expressed Sequence Tags are unique DNA sequences generated by sequencing cDNA clones from cDNA libraries
-
What are some problems with an EST approach of annotating the genome?
- 1. strong bias for abundant mRNA
- 2. miss-priming (incorrect 3' ends)
- 3. premature stop of the reverse transcriptase (incorrect 5' ends)
-
What is CAP biotinylation used for?
ensuring complete cDNA libraries constructed (rather than partial reads where the reverse transcriptase fell off)
-
Using biotin to make a complete cDNA library (picture):
-
Three ways you can sequence full length cDNAs (back in the ol' days)
- 1. Primer walking
- 2. transposon sequencing
- 3. Concatenation of cDNAs
-
Describe sequencing with transposons:
-
Describe sequencing via primer walking:
-
Some benefits of a full length cDNA library collection:
- Experimental annotation of the genome
- Uni-gene set of cDNA (microarrays)
- Systematic protein expression
- Systematic protein-protein interaction studies
- Systematic overexpression and antisense studies
-
Describe the Gateway System (Invitrogen):
- Gateway cloning is based on the highly specific integration and excision reactions of bacteriophage λ into and out of the Escherichia coli genome (Hartley et al. 2000). The Gateway cloning system uses two different enzyme mixtures, each of which performs a different type of recombination reaction.
-
-
Key methods of ENCODE
- DNaseq + FAIRE-seq + ATAC-sec: Open chromatin
- ChIP-seq: protein/DNA interactions
- RNA-seq: transcribed protein and non-coding RNAs
- RRBS: DNA methylation
- 5C: long range interactions between genomic regulatory regions
-
Which technique do you use when you have a protein, and you want to know which sections of DNA it interacts with?
- ChIP-seq
-
ChIP-seq vs. DAP-seq
ChIP-seq captures DNA associated TFs in vivo, whereas DAP-seq identities TF-DNA interactions in vitro
|
|
