Wednesday 23 September · 2020. 9. 23. · DTU Sundhedsteknologi Lab day 1: Purification Step 1 •...

Preview:

Citation preview

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

������Ȭ����¡�� ���������� �������� ��� ����¡ ���� ����� ��� ���� ŗǯ

������ ŗ ������Ȭ����¡�� ����������

����ȱŗ����ȱŗ ����� � ��� �������� ���� ŗ ���������� �������� ����� �������� �������� ����� ������ ��� ��ǯ ��� ���� ŗ ���������� ������ �� �������� �� ��� �������� ������������ ��� ������� ���������� ������� �� ��� ����ǯ` ���� ���������� ����Ȭ����¡�� ��¡���� ���������ǰ ¢�� ���� ��� ��� ���������������� �������� �� ��� ������ ���� ����¡ ���������� ������ ��¡ǰ �ŗŖǰ  ������ ���� �� ��� ���� �� ����� �� �Ŝǯ ��� ���� �����������ǰ ��� ��� ���� ���� ����ǰ���� Ǜ ŗśŖŖŜŗŜśǯ

` ���� ���������� ������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

Recommended