wednesday 23 september · 2020. 9. 23. · dtu sundhedsteknologi lab day 1: purification step 1 •...

DTU Sundhedsteknologi

Wednesday 23 September

1

What have we done in the lab??


Bioanalyser results final Libraries



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4


Group

Form groups with 3-4 person

https://docs.google.com/spreadsheets/d/1LunO90es6dBKfm26jPz7XcAhi2xuBBNWnNU_2EHSaP0/edit#gid=22555979

https://docs.google.com/spreadsheets/d/1LunO90es6dBKfm26jPz7XcAhi2xuBBNWnNU_2EHSaP0/edit


Microbiome

SampleDNA

Sequencing

Data Analysis Associations

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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

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Purification Step 1



Step 2-3


Step 4


Step 5


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Purification Step 1



Step 2-3


Step 4


Step 5


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Measure the concentration of DNA

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DNA Fragmentation byFocused-ultrasonicator

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Bioanalyser profile


Fragmentation


Lab day 2: Library preparation

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Step 1: DNA Fragmentation/ End repair

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Step 1: DNA Fragmentation/ End repair

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Adaptor: “ TruSeq –style” indexed adaptors19

5'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC

GCTCTTCCGATCT

CGAGAAGGCTAG

3’GTTCGTCTTCTGCCGTATGCTCTATGTAGCCACTGACCTCAAGTCTGCACA

T AA+


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


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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Single index

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6LQJOH�,QGH[HG

6HTXHQFLQJ

2YHUYLHZ

6HTXHQFLQJ'XDO�,QGH[HG/LEUDULHVRQ WKH +L6HT �

6LQJOH�,QGH[HG6HTXHQFLQJ2YHUYLHZ

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Barcoding and pooling

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Sample Sheet

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De-Multiplexing

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Clean up

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Size selection and clean-up using SPRI BeadsSPRI = Solid Phase Reversible Immobilization

Ratio of SPRI beads to sample determines size cut off


Purification

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50µL

dNTP

dNTP


AMPure Beads

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Size selection

35µL

12µL

Supernatant


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wednesday 23 september · 2020. 9. 23. · dtu sundhedsteknologi lab day 1: purification step 1 •...

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