Banana Slug Genomics

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===== Library Construction ======
{{:lecture_notes:235_library_construction.pdf|Nader's Powerpoint}} (Converted to PDF)
==== General Overview ====

Current library generation methods more or less follow this general set of steps.

  - Double stranded sample either cut up randomly or by some targeted approach.
  - The overhanging ends are either finished with polymerase (if on the right strand) or digested with exonuclease.
  - The blunt ended fragments are A tailed specifically on the 3' ends of the DNA.
  - Double stranded library adapters have overhanging T which allows ligation to blunt ends in a specific orientation.
    - This orientation may be used to ensure single stranded samples
 
==== 454 Specific Library Construction ====

==== SOLiD Specific Library Construction ====
Since you are dealing with a short sequence you need a larger quantity of material to start due to loss in the gell size selection phase.

=== Mate Pair Library ===

==== Illumina Specific LIbrary Construction ====

=== Paired End Library ===

==== Targeted RNA Amplification ====

Capable of amplifying with high specificity with low (~2%) ribosomal RNA contamination.

  - First Strand cDNA synthesis
  - Second strand cDNA synthesis
  - SPIA Amplification
    - RNAse H cuts up the RNA (SPIA) adapter because it is bound to the now DNA poly-A tail of the original RNA sample, and strips away some of one of the strands of cDNA randomly.
    - Anneal back on an RNA SPIA primer (with attached RNA sequence to re-bind to the DNA poly-A tail for starting over from the first part of this step)
    - Extend the cDNA sequence back out to its fullest extent with polymerase, adding the remainder of the partially degraded cDNA strand to the linearly growing pool of cDNA sample.
    - Add RNAse H and continue with these steps until desired quantity is achieved.
Banana Slug Genomics

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