Sequencing Methods and Purposes

transposable elements

mobile pieces of DNA that can copy themselves into entirely new areas

ATAC-seq

technique for assessing chromatin accessibility

ATAC-seq is a technique to sequence ___________ ___________ and accessible ___________

open chromatin, DNA

ATAC-Seq acronym

assay for transposase accessible chromatin followed by sequencing

Tn5 transposase binds ___________ DNA

accessible (less compact)

Tn5 transposase ___________ the ___________ DNA and adds ___________ to it

cleaves, accessible, adaptors

Tn5 transposase adding adaptor sequences is called

tagmentation

___________ follows fragmentation and tagmentation

purification, PCR, next gen sequencing, data analysis

peaks of FastQ file correspond to

open chromatin

degrees of chromatin phase seperation

active, repressed, inert

TADS

topologically associating domains

DNA crosslinking

formaldehyde is used to bind two strands of DNA close together near each other

purpose of Hi-C sequencing

analyze how close sequences are to each other in the space of the nucleus

topologically associating domains (TADs)

DNA sequences within a TAD physically interact with each other more frequently than with sequences outside the TAD

Steps of Hi-C sequencing

sample, crosslink chromatin, fragment chromatin using endonucleases, repair and biotinylate ends, ligate fragmented chromatin, shear DNA and pull down biotinylated DNA, paired end sequencing, bioinformatics

Hi-C results are a

contact map

step 1 of next generation sequencing

amplified DNA sample is fragmented

step 2 of next generation sequencing

oligonucleotide adapters are put on the ends of DNA fragments

step 3 of next generation sequencing

fragments are added to flow cell where adapters bind to the flow cell and polymerase chain reactions synthesize a copy of each fragment

step 4 of next generation sequencing

original fragments are washed away

step 5 of next generation sequencing

bridge amplification

step 6 of next generation sequencing

reverse strands are cleaved

step 7 of next generation sequencing

flourescently labeled nucleotides are added to the sample and bind their complements, a camera captures their excitation by a laser

one "read" represents

the sequence of a single fragment

alignment

matching each read to a reference sequence

coverage

how many times each spot in a genome is read

variant calling

detecting where the reads are different from the reference genome

areas where variant calls are less likely to be mutations

repetitive regions, places with low coverage, ends of reads (slippage of polymerase)