Genomics & NCBI Accessions

Question 0

Problems with sequencing

Answer 0

Data is not always perfect
Each region needs to be covered about 10 times
20% of the reactions fail
Ex. Takes 400 days to cover the human genome

Question 1

How many base pairs is the human genome?

Answer 1

3.3x10^9 bp

Question 2

What is the basis of whole genome shotgun sequencing?

Answer 2

Sequence first, map later
Genome is cut into small pieces and sequenced
Overlapping regions are put in order

Question 3

When does shotgun sequencing work well?

Answer 3

Small bacterial genomes that don’t have much repetitive DNA

When sequencing different individuals of the same species

Question 4

What is done when gaps are encounters in shotgun sequencing

Answer 4

PCR primers are made from both ends of the contigs that cover gal region
Gap is amplified by PCR
PCR product is sequenced directly
Works well for small gaps

Question 5

What is done for large gaps that can’t be bridged by PCR

Answer 5

Are cloned into a low copy clone vector like BAC with an F origin of replication
Each end of the BAC is sequenced-paired ends-until the gap is covered

Question 6

Why is repetitive DNA a problem for genome sequencing

Answer 6

If a repeat is sequenced, there is no way to tell which part of the genome it came from
Unique flanking region is not included in sequenced fragment
Ex: LINES- long repeat units
Not sure effective for many eukaryotes

Question 7

What is the basis of the ordered clone approach

Answer 7

Map first, sequence later

Question 8

How is map first, sequence later done?

Answer 8

Physical map that contains ordered clones is produced first
Large fragments of genomic DNA are cloned into BAC vectors
Determine if BAC have overlapping sequences
Pick a minimal tiling path: least number of BAC that cover a specific path
Each BAC is sequenced using shotgun approach
BAC sequences are assembled into long contigs

Question 9

What percent of the genome is transposable elements?

Answer 9

45%

Question 10

What percent of the genome is exons?

Answer 10

3%

Question 11

What was the purpose of the 1000 Genomes Project?

Answer 11

to find the extent of genome variation among individuals

Question 12

What is the ENCODE project doing?

Answer 12

• find which regions of the human genome are transcribed into RNA and bind to TF
• understand chromatin structure

Question 13

What is the goal of the ENCODE project?

Answer 13

to characterize all noncoding DNA in the genome

Question 14

Define transcriptome.

Answer 14

-where a gene is expressed

Question 15

Explain how RNA-seq is done.

Answer 15

1. RNA is isolated from a cell and converted to cDNA
2. sequenced through illumina high throughput sequencing

-number of reads is how much mRNA is present in the sample

Question 16

Why is RNA-seq a better technique?

Answer 16

directly sequences cDNA instead of waiting for sequences to hybridize equally

Question 17

What is the unit of RNA-seq?

Answer 17

RPKM

reads per kilobase of exon per million mapped reads

Question 18

What is a regulon?

Answer 18

-genes that are coregulated

Question 19

Describe how microarray is done.

Answer 19

1. PMs and MM are sequenced for each gene. (MM is a negative control and measure background binding)
2. correct cDNA will bind strongly to PM
3. PM and MM are synthesized on a silicon chip
4. image produced on microarray.

Question 20

What is the complement for 5’ ATG

Answer 20

TAC5’ in the complement strand

5ATG in the DNA