Sample submission guide for NGS Favourites

SAMPLE SUBMISSION GUIDE
Amplicon Sequencing Favourites
Uni- and bidirectional amplicon sequencing with GS Junior
Sending samples according to the requirements below helps us to do our job better
and provides you best possible data quality!
General Amplicon preparation requirements
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Amplicon length: 250 – 500 bp (including adaptors)
Each PCR in sample pool has to be tagged by unique multiplex identifier (MID)
For your convenience please find a list with 48 individual MID sequences for your successful
primer design and primer ordering at the end of this document
Please choose the following primer setup for each of your PCR samples:
1x forward primer A (= A-key primer)
5’‐CGTATCGCCTCCCTCGCGCCATCAG‐(MID)‐forward-specific-sequence-3’
1x reverse primer B (= B-key primer)
5’‐CTATGCGCCTTGCCAGCCCGCTCAG‐(MID)‐reverse-specific-sequence-3’
Unidirectional sequencing
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For unidirectional sequencing a MID is needed only on one side of the PCR product (= the
end of PCR that is to be sequenced - e.g. either select PCR A-side or PCR B-side (figure 1)
e.g.: Sample 1 should have MID #1 on forward primer A-side or reverse primer B-side,
sample 2 should have MID #2 on forward primer A-side or reverse primer B-side, etc.
Do not mix the location of MID within one pool of PCR
The sequencing direction of projects with unidirectional sequencing can be selected during
the online ordering process (step 3)
If you have already prepared PCR samples containing MID sequences on both ends, these
samples can be also be sequenced unidirectional from primer A-side or primer B-side
Adding a MID on both ends maintains flexibility for bidirectional sequencing at a later stage
Figure 1 - MID only on one side of the amplicon (sufficient for unidirectional sequencing)
Eurofins Genomics I Anzinger Str. 7a I 85560 Ebersberg I Germany I eurofinsgenomics.com I [email protected] I +49 8092 8289-0
Bidirectional sequencing
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For bidirectional sequencing a MID is needed on both sides of PCR product (figure 2)
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Depending on your individual sequencing needs, MID on forward-specific-sequence and
reverse-specific-sequence primer (e.g. A-key primer or B-key primer) may have identical or
different sequences
Figure 2 - MID on both sides of the amplicon (mandatory for bidirectional sequencing)
PCR pool requirements
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Please provide pooled PCR samples (1 tube per PCR pool)
Pooling is recommended to be done with PCR fragments of about same length. Please note
that shorter fragments often perform better in pools
Please deliver the DNA solved in TRIS buffer / buffer from commercial DNA extraction kits
Please use 1.5 – 2.0 ml safe-lock tubes for shipping
Use the concentrations and volumes below for each PCR sample pool to be sequenced:
Sample Type
without gel purification
with gel purification
Minimum amount of DNA
500 ng – 1 µg
1 – 2 µg
Concentration
10 – 20 ng/µl
20 – 40 ng/µl
Volume
> 25 µl
> 25 µl
If possible, please supply us with the 2-3 fold amount of PCR sample material.
Please quantify the DNA concentration with fluorometric methods. Concentration determination by spectrometric methods (UV
photometer) may deliver imprecise DNA concentration values.
Eurofins Genomics I Anzinger Str. 7a I 85560 Ebersberg I Germany I eurofinsgenomics.com I [email protected] I +49 8092 8289-0
Sample Submission
Delivery of sample material
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Correct sample labelling is in the responsibility of the customer
Eurofins MWG Operon would recommend delivery on dry ice
Please send your samples including a copy of your order confirmation from the online shop
Please send your samples to:
NGS - Laboratory
Eurofins Genomics
c/o Dr. Arman Tehrani – Dr. Elmar Schilling
Anzinger Str. 7a
85560 Ebersberg
Germany
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For Non-EU countries: Please follow the customs instructions, the customs documents must
state - purified DNA, not harmful, not infectious, for research only.
Delivery of additional information
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Complete primer sequences inclusive the specific primer sequences as well as a MID-tag list
will have to be sent as FASTA file/Excel-file prior to start of the sequencing process to your
sales representative or to the following Email-address: [email protected].
List of 48 individual MID sequences for bar-coded fusion primer:
MID-01
MID-02
MID-03
MID-04
MID-05
MID-06
MID-07
MID-08
MID-10
MID-11
MID-13
MID-14
MID-15
MID-16
MID-17
MID-18
ACGAGTGCGT
ACGCTCGACA
AGACGCACTC
AGCACTGTAG
ATCAGACACG
ATATCGCGAG
CGTGTCTCTA
CTCGCGTGTC
TCTCTATGCG
TGATACGTCT
CATAGTAGTG
CGAGAGATAC
ATACGACGTA
TCACGTACTA
CGTCTAGTAC
TCTACGTAGC
MID-19
MID-20
MID-21
MID-22
MID-23
MID-24
MID-25
MID-26
MID-27
MID-28
MID-29
MID-30
MID-31
MID-32
MID-33
MID-34
TGTACTACTC
ACGACTACAG
CGTAGACTAG
TACGAGTATG
TACTCTCGTG
TAGAGACGAG
TCGTCGCTCG
ACATACGCGT
ACGCGAGTAT
ACTACTATGT
ACTGTACAGT
AGACTATACT
AGCGTCGTCT
AGTACGCTAT
ATAGAGTACT
CACGCTACGT
MID-35
MID-36
MID-37
MID-38
MID-39
MID-40
MID-41
MID-42
MID-43
MID-44
MID-45
MID-46
MID-47
MID-48
MID-49
MID-50
CAGTAGACGT
CGACGTGACT
TACACACACT
TACACGTGAT
TACAGATCGT
TACGCTGTCT
TAGTGTAGAT
TCGATCACGT
TCGCACTAGT
TCTAGCGACT
TCTATACTAT
TGACGTATGT
TGTGAGTAGT
ACAGTATATA
ACGCGATCGA
ACTAGCAGTA
(Sequence of 10 bases that have a sequence-error optimized design)
More MID sequences are available if your experiment requires more than 48 unique MIDs.
Eurofins Genomics I Anzinger Str. 7a I 85560 Ebersberg I Germany I eurofinsgenomics.com I [email protected] I +49 8092 8289-0