wednesday 23 september · 2020. 9. 23. · dtu sundhedsteknologi lab day 1: purification step 1 •...
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DTU Sundhedsteknologi
Wednesday 23 September
1
What have we done in the lab??
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DTU Sundhedsteknologi
Bioanalyser results final Libraries
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DTU Sundhedsteknologi
Bioanalyser results final Libraries
3
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DTU Sundhedsteknologi
Bioanalyser results final Libraries
4
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DTU Sundhedsteknologi
Group
Form groups with 3-4 person
https://docs.google.com/spreadsheets/d/1LunO90es6dBKfm26jPz7XcAhi2xuBBNWnNU_2EHSaP0/edit#gid=22555979
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DTU Sundhedsteknologi
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DTU Sundhedsteknologi
Microbiome
SampleDNA
Sequencing
Data Analysis Associations
7
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DTU Sundhedsteknologi
Lab day 1: Purification Step 1
• Homogenization of the samples
• Protein precipitation (Sodium Dodecyl Sulfate (SDS))
Step 2-3
• Precipitate non-DNA organic and inorganic material including humic substances acid, cell debris, and proteins
Step 4
• Contain a high concentration salt solution, which ensures effective DNA binding
Step 5
• Washing step to clean the DNA
8
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DTU Sundhedsteknologi
Purification Step 1
• Homogenization of the samples
• Protein precipitation (Sodium Dodecyl Sulfate (SDS))
Step 2-3
• Precipitate non-DNA organic and inorganic material including humic substances acid, cell debris, and proteins
Step 4
• Contain a high concentration salt solution, which ensures effective DNA binding
Step 5
• Washing step to clean the DNA
9
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DTU Sundhedsteknologi
Purification Step 1
• Homogenization of the samples
• Protein precipitation (Sodium Dodecyl Sulfate (SDS))
Step 2-3
• Precipitate non-DNA organic and inorganic material including humic substances acid, cell debris, and proteins
Step 4
• Contain a high concentration salt solution, which ensures effective DNA binding
Step 5
• Washing step to clean the DNA
10
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DTU Sundhedsteknologi
Measure the concentration of DNA
11
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DTU Sundhedsteknologi
DNA Fragmentation byFocused-ultrasonicator
12
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DTU Sundhedsteknologi
Bioanalyser profile
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DTU Sundhedsteknologi
Fragmentation
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DTU Sundhedsteknologi
Fragmentation
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DTU Sundhedsteknologi
Lab day 2: Library preparation
16
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DTU Sundhedsteknologi
Step 1: DNA Fragmentation/ End repair
17
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DTU Sundhedsteknologi
Step 1: DNA Fragmentation/ End repair
18
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DTU Sundhedsteknologi
Adaptor: “ TruSeq –style” indexed adaptors19
5'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC
GCTCTTCCGATCT
CGAGAAGGCTAG
3’GTTCGTCTTCTGCCGTATGCTCTATGTAGCCACTGACCTCAAGTCTGCACA
T AA+
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DTU Sundhedsteknologi
T AA
Fragmented DNA
+
Adaptors“TruSeq –style” indexed
adaptors
Advantages:
Index independent of read
-> more data
-> no more clustering
problems
Problems:
Need more reagents
Index only on one side
TA
AT
TA
AT
Forward read
Reverse read
Index read
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DTU Sundhedsteknologi
Cluster 21
Clusters in a contained
Environment (no need for clean rooms)
Sequencing performed in the flow cell on the clusters
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DTU Sundhedsteknologi
22
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DTU Sundhedsteknologi
Single index
23
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����ȱŘ ���¢���������� ����¡ ���� ������� �� ������� ��� ��� �������� �������� ������ �� ���� ������������ ��� ������������¢ ������ǯ ����ǰ ��� �������� �������� ������ �� �������
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DTU Sundhedsteknologi
Barcoding and pooling
24
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DTU Sundhedsteknologi
Sample Sheet
25
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DTU Sundhedsteknologi
De-Multiplexing
26
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DTU Sundhedsteknologi
Clean up
27
Size selection and clean-up using SPRI BeadsSPRI = Solid Phase Reversible Immobilization
Ratio of SPRI beads to sample determines size cut off
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DTU Sundhedsteknologi
Purification
28
50µL
dNTP
dNTP
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DTU Sundhedsteknologi
AMPure Beads
29
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DTU Sundhedsteknologi
Size selection
35µL
12µL
Supernatant
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DTU Sundhedsteknologi
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