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Next-Gen Sequencing Sample
Specification and Submission Guideline
1. DNA Sequencing Application -------------------------------------------------------------------- 2
1.1. DNA Sample Quality and Quantity Specification
1.2. Summary of DNA Sample Specification
2. RNA Sequencing Application --------------------------------------------------------------------5
2.1. RNA Sample Quality and Quantity Specification
2.2. Summary of RNA Sample Specification
3. Pre-made Libraries Specification
--------------------------------------------------------7
4. Sample Submission-------------------------------------------------------------------------------- 7
4.1. DNA Samples
4.2. RNA Samples
4.3. Sample Shipping
4.4. Checklist
NGS Sample specification and submission guideline
Macrogen Inc.
V13.01 July 2013
1
1. DNA Sequencing Application
The conditions of the starting DNA are very important to achieve meaningful data. We
promise to deliver the best possible outcome but at the same time, we request our
clients to meet the minimum sample specification requirements as indicated below.
1.1 DNA Sample Quality and Quantity Specification

The ratio of OD260/280 is used as an indicator of sample purity where the
values of 1.8–2.0 are an indication of a good DNA sample. However, this
measurement can be compromised by the presence of RNA or small nucleic
acid fragments such as nucleotides. Thus, gDNA samples should be carefully
collected to ensure that they are free of any contaminants.

Gel electrophoresis is a powerful means for revealing the condition, including
the actual presence or absence of DNA in a sample. Impurities, such as
detergents or proteins, are typically revealed by the presence of smeared
DNA bands. The interferences of RNA at 260 nm readings are often visible at
the bottom of a gel. A ladder or smear below a band of interest may
indicatethe nicking or other damage to the DNA. Where possible, a gel
electrophoresis should be performed to assess the condition of the DNA
sample (Figure 1).
<Figure1. An example of acceptable gDNA in GE>
1
2
3
4
1% agarose gel, 30min running at 120V
lane 1: gDNA, lane2:1kb ladder, lane 3: 100ng conc. Marker, lane4:50ng conc. Marker

The correct quantification of gDNA is essential in library construction.
Macrogen recommends quantifying the starting genomic material by using a
fluorescence‐based quantification method to accurately measure the quantity
of dsDNA.
NGS Sample specification and submission guideline
Macrogen Inc.
V13.01 July 2013
2
1.2 Summary of DNA sample specification
Note: The only most commonly used library types are listed below. If your
samples do not comply with this guidelines, please contact us and we can
discuss your specific sample requirements.
Library type
QC Criteria
Conc.
Purity
Volume
Total Amount
(ng/㎕)
(A260/A280)
(㎕)
(㎍)
TruSeq DNA library
>60ng/㎕
>1.7
>20㎕
>1.2㎍
Mate paired End library (2~5kb)
>100ng/㎕
>1.7
>100㎕
>10㎍
Mate paired End library (6~10kb)
>200ng/㎕
>1.7
>100㎕
>20㎍
Mate paired End library (11~20kb)
>400ng/㎕
>1.7
>100㎕
>40㎍
Nextera Mate Pair (Gel-free)
> 20 ng/㎕
> 1.7
> 50 ㎕
>1 ㎍
Nextera Mate Pair (Gel-plus)
> 100 ng/㎕
> 1.7
> 40 ㎕
>4 ㎍
Truseq DNA PCR-free (350bp insert)
> 20 ng/㎕
> 1.7
> 50 ㎕
>1 ㎍
Truseq DNA PCR-free (550bp insert)
> 20 ng/㎕
> 1.7
> 100 ㎕
>2 ㎍
Nimblegen EZ Whole Exome
>60ng/㎕
>1.7
>20㎕
>1.2㎍
Nimblegen Array
>60ng/㎕
>1.7
>20㎕
>1.2㎍
Sureselect_Human 50Mb
>50ng/㎕
>1.7
>20㎕
>1.0㎍
Sureselect_Mouse 50Mb
>50ng/㎕
>1.7
>20㎕
>1.0㎍
Sureselect_Kinome
>50ng/㎕
>1.7
>20㎕
>1.0㎍
Sureselect_V4_PostCap
>50ng/㎕
>1.7
>20㎕
>1.0㎍
Sureselect_V4+UTR_PostCap
>50ng/㎕
>1.7
>20㎕
>1.0㎍
Sureselect_V4,_PreCap
>50ng/㎕
>1.7
>20㎕
>1.0㎍
Sureselect_V4+UTR_PreCap
>50ng/㎕
>1.7
>20㎕
>1.0㎍
TruSeq Exome Enrichment
>60ng/㎕
>1.7
>20㎕
>1.2㎍
Nextera Rapid exome capture
>2.5ng/㎕
>1.7
>20㎕
>0.05㎍
[HiSeq 2000/2500]
ChIP seq library*
>1.7
>0.01㎍
Nextera library
>2.5ng/㎕
>1.7
>20㎕
>0.05㎍
Nextera XT library
>0.2ng/㎕
>1.7
>5㎕
>0.001㎍
TruSeq HT library (dual indexing)
>50ng/㎕
>1.7
>20㎕
>1.0㎍
Whole Genome Bisulfite treated library
>100ng/㎕
>1.7
>50㎕
>5.0㎍
MBD enriched library
>20ng/㎕
>1.7
>50㎕
>1.0㎍
Amplicon library
> 20 ng/ul
> 1.7
> 10ul
> 0.2ug
*Macrogen use DNA 1000 / 7500 chip for normal PCR product, high sensitivity chip for very small amount of DNA fragment such as ChIPed DNA.
NGS Sample specification and submission guideline
Macrogen Inc.
V13.01 July 2013
3
Library type
QC Criteria
Conc.
Purity
Vol.
Total Amount
(ng/㎕)
(A260/A280)
(㎕)
(㎍)
RL library (>1.5kb, for FLX+)
>50ng/㎕
>1.7
>20 ㎕
>1.0 ㎍
RL library(>1.5kb, for GS-FLX)
>25ng/㎕
>1.7
>20 ㎕
>0.5 ㎍
PCR product (LMW)
>20ng/㎕
>1.7
>10 ㎕
>0.2 ㎍
Amplicon library
>10ng/㎕
>1.7
>20 ㎕
>0.2 ㎍
Mate paired End library (3kb)
>100ng/㎕
>1.7
>50 ㎕
>5.0 ㎍
Mate paired End library (8kb)
>100ng/㎕
>1.7
>150 ㎕
>15 ㎍
Mate paired End library (20kb)
>100ng/㎕
>1.7
>300 ㎕
>30 ㎍
[GS-FLX/ FLX+]
NGS Sample specification and submission guideline
Macrogen Inc.
V13.01 July 2013
4
2. RNA Sequencing Application
Preventing RNase contamination is a mandatory requirement when handling RNA
samples. We request our clients to follow the commercial or certified in-house protocols
to minimize any risks in sample degradation.
2.1 RNA Sample Quality and Quantity Specification

The use of degraded RNA for NGS applications can result in low yield, over‐
representation of the 5’ ends of the RNA molecules, or failure of the library
construction. We recommend the use of Agilent Technologies 2100
Bioanalyzer to check the total RNA integrity. The specification for RNA
Integrity Number (RIN) value of greater than or equal to 8 is required. We
also request our clients to perform a DNase treatment along with the RNA
isolation method prior to the sample shipment.

As part of our initial quality control, we primarily use Agilent Technologies
2100 Bioanalyzer to determine the size, purity, and concentration of the
sample and further evaluation may be done by performing gel electrophoresis.
A denaturing 1% agarose gel can be run against a 1 kb ladder and the RNA
integrity assessed upon staining with ethidium bromide. High-quality RNA
shows a 28S rRNA band at 4.5 kb of twice the intensity of the 18S rRNA band
at 1.9 kb. Messenger RNA appears as a faint smear from 0.1 to 12 kb
(Figure 2).
<Figure 2.An example of Acceptable RNA QC>
< 2100 Bioanalzyer RNA Nano Chip>
NGS Sample specification and submission guideline
Macrogen Inc.
V13.01 July 2013
5
2.2 Summary of RNA sample specification
QC Criteria
Library type
[GS-FLX/ FLX+]
Status
cDNA Rapid library
Total RNA
cDNA Rapid library
purified
mRNA
Conc.
Vol.
Total Amount
(ng/㎕)
(㎕)
(㎍)
>250ng/㎕
200 ㎕
>50 ㎍
>11ng/㎕
>19 ㎕
>0.2 ㎍
RIN
rRNA
ratio
>7.0
>1.5
>7.0
>1.5
>7.0
>1.5
>7.0
>1.5
Library type
[HiSeq 2000/2500]
mRNA library
total RNA
>1.0 ㎍
mRNA library
mRNA
>0.1 ㎍
Strand-specific RNA library
total RNA
>4.0 ㎍
Strand-specific RNA library
mRNA
>0.1 ㎍
small RNA library
total RNA
>3.0 ㎍
all small RNA
small RNA library
mRNA
from 1~10㎍ of
total RNA
NGS Sample specification and submission guideline
Macrogen Inc.
V13.01 July 2013
6
3. Pre-made Libraries Specification
Pre-made libraries can be submitted only after a consultation was made with our team
members. Below are the general specifications:

Requirements:
Volume: 20ul
Concentration (nM): 5
Concentration (ng/㎕): >1.5
Size: 400~500bp
OD260/280 ratio: 1.8 and 2.0

Evaluate library concentration, size and quality (Please provide us the QC
result)

Short-insert libraries (160-180bp): minimum 3㎍

Index/MID information is required if applicable.
4. Sample Submission
4.1 DNA Samples

Submit DNA sample(s) in Distilled water or Elution bufferat a minimum
concentration of 100ng/㎕ in a clearly labeled 1.5~2.0ml microcentrifuge tube
tightly sealed and parafilm wrapped.

Include a brief description of the DNA extraction and/or enrichment protocol
used.

Can be delivered in an ambient temperature or with ice pack.
4.2 RNA Samples

Submit RNA sample(s)in DEPC treated water, or EtOHin a clearly labeled
1.5~2.0ml microcentrifuge tube tightly sealed and parafilm wrapped.

Include a brief description of the RNA extraction and/or enrichment protocol
used.

Dry ice shipping: We recommend using a minimum of 3-5kg of dry ice which
should last for 24-48hrs.
NGS Sample specification and submission guideline
Macrogen Inc.
V13.01 July 2013
7
4.3 Sample Shipping

Place the sealed microcentrifuge tubes in a 50ml disposable screw cap tube
or other suitable container for an additional insulation during shipment to
minimize any risk of physical damages during shipment. Additionally, cotton
and absorbent paper can be used to hold the containers in place.


You should include the followings in the package.
-
Prepared Sample with ice pack or dry ice.
-
NGS project Order Sheet (with QC files, if applicable)
-
Client Information
Ship samples to:
Macrogen Inc. (NGS Division)
10F World Meridian Venture Center
#60-24 Gasan-dong Geumchun-gu
Seoul 153-781 South Korea
Phone +82-2-2113-7352 (Attn. Jiyoung Kim)
4.4 Quick Sample Submission Checklist
□ OrderSheet template and guideline received by e-mail.
□ Gel photo and NanoDrop measurement result (amount and OD260/280 ratio).
□ DNA samples quantified using Picogreen method (optional).
□ Agilent 2100 Bioanalyzer data collected(optional).
□ All Samples tightly sealed, spun down and/or frozen.
□ Include the ordersheet prepared in the sample box.
NGS Sample specification and submission guideline
Macrogen Inc.
V13.01 July 2013
8