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--- archive:lab_protocols:illumina_run1 [2010/04/28 18:11]
hyjkim
+++ archive:lab_protocols:illumina_run1 [2015/09/02 16:57] (current)
ceisenhart ↷ Page moved from lab_protocols:illumina_run1 to archive:lab_protocols:illumina_run1
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-Overview: This sample was prepared according to manufacturer's recommendations.
+Overview: This sample was prepared according to manufacturer's recommendations. [Add link to illumina protocol]
-==== DNA Extraction ====
+==== Starting DNA ====
-==== Library Preparation ====
+==== Initial Size Selection ====
-==== PCR ====
+{{:lab_protocols:bana_slug_library_illumina.jpg?600}}
+{{:lab_protocols:bana_slug_library_illumina._after_cutjpg.jpg.jpg?600}}
+[[http://www.fermentas.com/en/products/all/dna-electrophoresis/generuler-dna-ladders/sm0373-generuler-50bp|Fermentas O'generuler 50bp ladder used in these gels.]] Lanes 1 & 6
+µg of 98.0 ng/µl genomic DNA was sheared using either Covaris or a Nebulizer.
+Lanes 3&4: Banana Slug DNA sheared using Covaris.
+Lanes 5&6: Banana Slug DNA sheared using a Nebulizer. Both were cut between 250-375 basepairs.
+==== Bioanalyzer results of initial shearing and final amplified library size selection ====
+{{:lab_protocols:bioanalyzer-illumina-library.pdf|Bioanalyzer results of shearing distributions and final amplified libraries.}} Covaris and Nebulizer shearing are in lanes 1 and 2 respectively. Amplified and size selected libraries for Nebulizer and Covaris in lanes 3 and 4 respectively.
+The results indicate the final nebulizer library ranges from 202-320bp in distribution. The final covaris library ranges from 225-263bp in length.
+==== Final distribution of paired end reads ====
+Each stage indicates a different distribution in the length of the paired-end reads.
+The original size selection cut indicates a fragment size of 250-375. However, the bioanalyzer results indicate the final size selected fragment is much shorter at 200-320 bp for a final distribution between 100-230 bp.
+==== Differences between lanes in illumina run ====
+Each lane contained a different concentration of starting material used in bridge amplification.
+^Lane ^ Starting amount of library and library type ^
+|Lane 1 | 8 picoMoles Nebulized Library |
+|Lane 2 | 10 picoMoles Nebulized Library |
+|Lane 3 | 12 picoMoles Nebulized Library |
+|Lane 4 | 6 picoMoles phix control* |
+|Lane 5 | 8 picoMoles Covaris Library |
+|Lane 6 | 10 picoMoles Covaris Library |
+|Lane 7 | 12 picoMoles Covaris Library |
+|Lane 8 | 14 picoMoles Covaris Library |
+These different starting quantities of DNA are reflected in the number of clusters generated and the total reads outputted for each lane. Additionally, a different distribution of distance for the paired-end reads are different for lanes 1-3 and 5-8
+*Data from lane 4 of the illumina run **SHOULD NOT** be used for assembly
-==== Sequencing Data ====

Banana Slug Genomics

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