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archive:lab_protocols:illumina_run1

illumina Sequencing Run 1

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Prepared by Akram Tariq Sial

Date:

Overview: This sample was prepared according to manufacturer's recommendations. [Add link to illumina protocol]

Starting DNA

Initial Size Selection

bana_slug_library_illumina.jpg bana_slug_library_illumina._after_cutjpg.jpg.jpg

Fermentas O'generuler 50bp ladder used in these gels. Lanes 1 & 6

1µg 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

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

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archive/lab_protocols/illumina_run1.txt · Last modified: 2015/09/02 09:57 by ceisenhart