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archive:lab_protocols:illumina_run1 [2010/03/29 22:03] hyjkim created |
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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- | ===== Illumina Sequencing Run 1 ===== | + | ===== illumina Sequencing Run 1 ===== |
- | Overview: | + | ==== Meta ==== |
+ | Prepared by Akram Tariq Sial | ||
- | ==== DNA Extraction ==== | + | Date: |
- | ==== Library Preparation ==== | + | Overview: This sample was prepared according to manufacturer's recommendations. [Add link to illumina protocol] |
- | ==== Emulsion PCR ==== | + | ==== Starting DNA ==== |
+ | |||
+ | ==== Initial Size Selection ==== | ||
+ | |||
+ | {{: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 | ||
+ | |||
+ | 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 ==== | ||
+ | {{: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 ==== |