Genome Sequencing and Assembly Strategies

Flashcards covering hierarchical versus shotgun genome sequencing, repetitive genomic elements (SINEs, LINEs, satellite DNA), mate-pair genome assembly, quality control steps, and Sanger sequencing specifications.

Hierarchical Sequencing

A "top-down" genome sequencing approach where the genome is first physically mapped, fragmented into large chunks ($10\text{--}200\,\text{kb}$), cloned into Bacterial Artificial Chromosomes (BACs) or Yeast Artificial Chromosomes (YACs), subcloned into M13 vectors, sequenced, and assembled using the physical map.

Shotgun Sequencing

A "bottom-up" sequencing method where the entire genome is randomly broken into small pieces without prior physical mapping, sequenced from both ends, and computationally assembled by finding overlapping reads.

Physical Map

A genomic map that identifies the positions and minimal overlapping tiling paths of large cloned fragments, used in hierarchical sequencing to guide the assembly of sequenced clones.

Bacterial Artificial Chromosome (BAC)

A cloning vector used in hierarchical sequencing to maintain and propagate large genomic DNA fragments ($10\text{--}200\,\text{kb}$) in bacterial cells.

M13 Vector

A vector into which large BAC clones are subcloned into smaller fragments for individual sequencing in hierarchical sequencing workflows.

SINEs (Short Interspersed Nuclear Elements)

Short non-coding repetitive DNA sequences ($\sim 285\,\text{bp}$) that represent cDNA copies of RNA (retrosequences); they act as active transposons and account for approximately $13.14\%$ of the human genome.

Alu Element

The predominant SINE family in humans ($\sim 285\,\text{bp}$), named for containing an AluI restriction enzyme recognition site, present in roughly $1{,}090{,}000$ copies and making up $10.60\%$ of the human genome.

7SL RNA

A cellular RNA molecule identified as the evolutionary ancestor of human SINE elements, sharing significant sequence alignment and homology with the Alu consensus sequence.

LINEs (Long Interspersed Nuclear Elements)

Autonomous retroposon elements ($1\text{--}6\,\text{kb}$ in length, present in $868{,}000$ copies) that replicate through an RNA intermediate and encode their own reverse transcriptase enzyme.

L1 Element

The only member of the human LINE families (L1, L2, L3) that is still transpositionally active in humans, sharing homology with LINEs found in marsupials.

Satellite DNA

Tandemly repeated DNA sequences with repeat units ranging from $5\text{ to }300\,\text{bp}$ repeated $10^5\text{ to }10^6$ times, typically localized to heterochromatic regions such as centromeres and telomeres.

Human $\alpha$ Satellite DNA

A centromeric tandem repeat organized as $171\,\text{bp}$ monomers, dimers ($342\,\text{bp}$), or higher multimeric arrays (up to 16-mers), estimated to exist in approximately $400{,}000$ copies.

Human $\beta$ Satellite DNA

A pericentromeric repeat consisting of a $68\,\text{bp}$ monomer unit present in $30{,}000\text{--}60{,}000$ copies on metacentric chromosomes and acrocentric chromosomes 13, 14, 15, 21, and 22.

Mate Pairs

Sequencing reads generated from both opposing ends of a cloned DNA insert of a defined length ($8\text{--}10\,\text{kb}$), providing known physical distance and orientation to help resolve repetitive elements during genome assembly.

Contig

A continuous, consensus sequence formed by assembling overlapping sequencing reads.

Scaffold

A higher-order genomic assembly structure formed by linking, ordering, and orienting multiple contigs across gaps using mate-pair connections.

Rock (Genome Assembly)

An assembly element or contig connection that is supported and validated by two or more mate pairs.

Stone (Genome Assembly)

An assembly element or contig connection that is supported by only a single mate pair.

PHRED Score

A logarithmic metric used to estimate the accuracy and statistical probability of error for each base call in DNA sequencing data.

Purine Stacking

Strong base-stacking interactions between adjacent adenine (A) and guanine (G) residues along DNA or RNA strands, causing mobility compressions and sequencing artifacts in Sanger sequencing.

Sanger Sequencing Specifications

Sequencing characteristics including read lengths of $0.6\text{ to }1.5\,\text{kb}$, throughput of $1 \times 16$ (one sequence per capillary), forward or reverse single-direction sequencing, and a cost of \4\text{ to }\$5$ per sample.

Sequence Trimming and Preprocessing

The protocol of quality control prior to assembly that removes primer and vector sequences, masks repeat elements (LINEs, SINEs, satellite DNA), addresses ambiguous N's, and trims low-quality terminal bases.