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DTU Sundhedsteknologi
Wednesday 23 September
1
What have we done in the lab??
DTU Sundhedsteknologi
Bioanalyser results final Libraries
DTU Sundhedsteknologi
Bioanalyser results final Libraries
3
DTU Sundhedsteknologi
Bioanalyser results final Libraries
4
DTU Sundhedsteknologi
Group
Form groups with 3-4 person
https://docs.google.com/spreadsheets/d/1LunO90es6dBKfm26jPz7XcAhi2xuBBNWnNU_2EHSaP0/edit#gid=22555979
DTU Sundhedsteknologi
DTU Sundhedsteknologi
Microbiome
SampleDNA
Sequencing
Data Analysis Associations
7
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
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
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
DTU Sundhedsteknologi
Measure the concentration of DNA
11
DTU Sundhedsteknologi
DNA Fragmentation byFocused-ultrasonicator
12
DTU Sundhedsteknologi
Bioanalyser profile
DTU Sundhedsteknologi
Fragmentation
DTU Sundhedsteknologi
Fragmentation
DTU Sundhedsteknologi
Lab day 2: Library preparation
16
DTU Sundhedsteknologi
Step 1: DNA Fragmentation/ End repair
17
DTU Sundhedsteknologi
Step 1: DNA Fragmentation/ End repair
18
DTU Sundhedsteknologi
Adaptor: “ TruSeq –style” indexed adaptors19
5'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC
GCTCTTCCGATCT
CGAGAAGGCTAG
3’GTTCGTCTTCTGCCGTATGCTCTATGTAGCCACTGACCTCAAGTCTGCACA
T AA+
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
DTU Sundhedsteknologi
Cluster 21
Clusters in a contained
Environment (no need for clean rooms)
Sequencing performed in the flow cell on the clusters
DTU Sundhedsteknologi
22
DTU Sundhedsteknologi
Single index
23
6LQJOH�,QGH[HG
6HTXHQFLQJ
2YHUYLHZ
6HTXHQFLQJ'XDO�,QGH[HG/LEUDULHVRQ WKH +L6HT �
6LQJOH�,QGH[HG6HTXHQFLQJ2YHUYLHZ
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` ���� ���������� ������Ȭ����¡�� ������ ��Ȧ�Ř ��������� �� ����Ȭ����¡�� ������� ���������ǰ ¢�� ��� ��� ������ �Ŝ �� �ŗŖǯ
����¡ ���� �������������� ����ȱŗ ������� �� ������� ��� ��� ����¡ ŗ ǻ�ŝǼ ���������� ������ �� �������� ����� ���� �������� ������ǰ ��������� ��� ����¡ ŗ ǻ�ŝǼ ����ǯ` ���� ���������� ����Ȭ����¡�� ��¡���� ���������ǰ ¢�� ���� ��� ��� ��������������������� ������ �������� �� ��� ������ ���� ����¡ ���������� ������ ��¡ǰ�ŗŘǰ ���� �� ���� �� ����� �� �Şǯ
` ���� ���������� ������Ȭ����¡�� ������ ��Ȧ�Ř ��������� �� ����Ȭ����¡�� ������� ���������ǰ ¢�� ��� ��� ������ �Ş �� �ŗŘǯ
����¡ ŗ ǻ�ŝǼ ��������� ��� ����¡ ���� �����������ǰ ��� ����¡ ŗ ǻ�ŝǼ ���� �� ���������ǯ` ���� ���������� ������Ȭ����¡�� ���������ǰ ��� ����¡ ���� �������� �� ����� �¢������ ����������ǯ
` ���� ���������� ����Ȭ����¡�� ��������� ǻ��¡���� �� ������ �Ǽǰ ��� ����¡ ������������ �� ����� �¢���� �� ����������ǯ
����ȱŘ ���¢���������� ����¡ ���� ������� �� ������� ��� ��� �������� �������� ������ �� ���� ������������ ��� ������������¢ ������ǯ ����ǰ ��� �������� �������� ������ �� �������
DTU Sundhedsteknologi
Barcoding and pooling
24
DTU Sundhedsteknologi
Sample Sheet
25
DTU Sundhedsteknologi
De-Multiplexing
26
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
DTU Sundhedsteknologi
Purification
28
50µL
dNTP
dNTP
DTU Sundhedsteknologi
AMPure Beads
29
DTU Sundhedsteknologi
Size selection
35µL
12µL
Supernatant
DTU Sundhedsteknologi
31