INSTRUCTION MANUAL
ACCEL-AMPLICON™ PANELS
Targeted NGS Amplicon Library Prep for Illumina® Platforms
Cat. No. AL-IL56G-12/48, 56G Oncology Panel
Cat. No. AL-ILTP53-12/48, Comprehensive TP53 Panel
Cat. No. AL-ILEGFR-12/48, EGFR Pathway Panel
Cat. No. AL-ILSID-48, Sample_ID Panel
www.swiftbiosci.com
Revision 160308
CONTENTS
Revision History .................................................................................................................................................................... 1
Experienced User Protocol ................................................................................................................................................. 2
Introduction............................................................................................................................................................................ 3
Protocol Overview ................................................................................................................................................................ 4
Before You Start ................................................................................................................................................................... 5
Quantifying Starting Input Material .................................................................................................................................... 6
FFPE .................................................................................................................................................................................. 6
Circulating, Cell-free Dna (cfDNA) ................................................................................................................................. 6
High Quality Genomic DNA, Whole Blood, and Fresh Frozen Tissue ..................................................................... 6
Prepare the Library .............................................................................................................................................................. 7
Input Quantification .......................................................................................................................................................... 7
Multiplex PCR Step .......................................................................................................................................................... 8
SPRI Step 1....................................................................................................................................................................... 8
Indexing Step .................................................................................................................................................................... 9
SPRI Step 2....................................................................................................................................................................... 9
Library Quantification ....................................................................................................................................................... 9
Bioinformatics Options..................................................................................................................................................... 9
Appendix A: Panel-Specific Information ......................................................................................................................... 10
56G Oncology Panel ...................................................................................................................................................... 10
Comprehensive TP53 Panel......................................................................................................................................... 11
EGFR Pathway Panel .................................................................................................................................................... 12
Sample_ID Panel ........................................................................................................................................................... 13
Appendix B: Accel-Amplicon Supporting Information ................................................................................................... 14
ALU115-qPCR Input Quantification Assay ................................................................................................................. 14
Sample Sheet – Special Considerations .................................................................................................................... 14
MiSeq Loading Recommendations.............................................................................................................................. 14
Troubleshooting Common Problems ........................................................................................................................... 15
Indexed Adapter Sequences ........................................................................................................................................ 16
General Warranty ........................................................................................................................................................... 17
Limitation of Liability....................................................................................................................................................... 17
Notice to Purchaser: Limited License.......................................................................................................................... 17
www.swiftbiosci.com
REVISION HISTORY
REVISION
DATE
DESCRIPTION OF CHANGE
160308
March 2016

Updated new Company logo and colorization.
160202
February 2016

Updated recommended PCR cycles for EGFR Pathway Panel.
151209
December 2015



Added EGFR Pathway Panel and Sample_ID Panel information.
Created Appendix A for panel-specific information.
Annotated headers and specific sections for “Quantifying Starting Input
Material” section for specific sample types.
Incorporated Troubleshooting Common Problems section.

www.swiftbiosci.com
1
EXPERIENCED USER PROTOCOL
Input Quantification
Use a qPCR-based assay for FFPE or cfDNA. Qubit® may be used only for high quality DNA samples.
Multiplex PCR Step
Assemble on ice
COMPONENT
VOLUME
Reagent G1*
Reagent G2
Enzyme G3
Sample DNA
Total
2 μl
3 μl
15 μl
10 µl
30 µl
MULTIPLEX PCR THERMOCYCLER PROGRAM
30 sec
10 sec
5 min
1 min
10 sec
98 °C
98 °C
63 °C
65 °C
98 °C
1 min
64 °C
1 min
Hold
65 °C
4 °C
4 cycles
21 cycles (56G)
22 cycles (TP53, Sample_ID)
23 cycles (EGFR Pathway)
*Reagent G1 is the Panel-specific set of multiplex amplification primers.
SPRI Step 1
Move samples to a separate post-PCR area before opening.
SAMPLE VOLUME
SPRI BEAD VOLUME
ELUTION VOLUME
30 µl
36 µl (ratio: 1.2)
Air-dry the pellet briefly. Proceed to the Indexing Step for
resuspension without delay.
Indexing Step
1. Add a unique combination of 5 µl Index D50X + 10 µl Index D7XX to each sample bead pellet.
2. Add 35 μl of the Indexing Reaction Mix below to each sample and resuspend the pellet (total volume 50 µl).
Incubate for 20 minutes at 37 °C (with lid heating OFF).
COMPONENT
VOLUME
Buffer Y1
Enzyme Y2
Enzyme Y3
Enzyme Y4
Total
31 μl
1 μl
1 μl
2 μl
35 µl
SPRI Step 2
SAMPLE VOLUME
PEG NaCl VOLUME
ELUTION VOLUME
50 µl
42.5 µl (ratio: 0.85)
20 µl (Post-PCR TE)
Library Quantification
Quantify a 1:100,000 dilution of the library in triplicate using a qPCR-based assay based upon a library size of 265 bp.
www.swiftbiosci.com
2
INTRODUCTION
Accel-Amplicon Panels for Illumina platforms enable the preparation of high quality targeted Next Generation Sequencing
(NGS) libraries from a variety of sample types, including formalin-fixed, paraffin-embedded (FFPE) and circulating, cellfree DNA (cfDNA). Adapters are included for dual indexing and multiplexing up to 96 samples on a sequencing run.
The kit utilizes Illumina-compatible adapter sequences and has been validated on Illumina platforms. The table below lists
key characteristics and typical performance of available panels using high quality control genomic DNA.
FEATURE
ACCEL-AMPLICON PANEL SPECIFICATION
Packaging Options
12/48 reactions with dual indexing
Input DNA Required*
10-25 ng
Time Required
2 hours
Amplicon Size
Average 140 bp (panel-dependent)
FFPE/cfDNA Compatible
Yes
Percent On Target
> 95%
Coverage Uniformity
(> 20% of Mean)
> 95%
*As quantified by qPCR. Please see section on Quantifying Starting Input Material. Qubit represents amplifiable DNA content more
accurately than NanoDrop, however is not as accurate as the qPCR assay. For sample types with more consistent high quality DNA
including whole blood, fresh frozen samples, and cultured cells, quantification by Qubit is a reliable indicator of amplifiable content.
www.swiftbiosci.com
3
PROTOCOL OVERVIEW


This protocol contains a Multiplex PCR step for the simultaneous production of hundreds of amplicon targets in a
single tube and an Indexing step for the addition of dual indexed adapters, enabling multiplexing of up to 96 unique
libraries.
Bead-based SPRI clean-ups are used to purify the sample by removing unused oligonucleotides and changing buffer
composition between steps.
www.swiftbiosci.com
4
BEFORE YOU START



Upon receipt, store the kit at -20 ºC.
Separate the Multiplex PCR Reagents (keep in pre-PCR area) and Indexing Reagents (keep in post-PCR area).
Please read this manual carefully before starting.
Kit Contents
Kit contains enough reagents for the preparation of either 12 or 48 libraries (10% excess volume provided).
Reagent G1*
12
REACTIONS
26 µl
48
REACTIONS
106 µl
Reagent G2
40 µl
160 µl
Index D7XX
Reagent G3
Pre-PCR TE
198 µl
1200 µl
800 µl
1200 µl
Buffer Y1
Enzyme Y2
Enzyme Y3
Enzyme Y4
Post-PCR
TE
KIT
REAGENTS
Multiplex
PCR
Reagents
KIT
REAGENTS
Indexing
Reagents
Index D50X
12
REACTIONS
11 µl each of
D501-D506
66 µl each of
D701-D702
410 µl
13 µl
13 µl
26 µl
1200 µl
48
REACTIONS
33 µl each of
D501-D508
44 µl each of
D701-D712
1637 µl
53 µl
53 µl
106 µl
1200 µl
*Reagent G1 is the Panel-specific set of multiplex amplification primers.
Required Materials Not Supplied












SPRIselect beads (Beckman Coulter, Cat. No. B23317/B23318/B23319)
PEG-8000 (Sigma-Aldrich, Cat. No. P5413) and NaCl (JT Baker, Cat. No. 3624-19) (see pg. 7 for recipe), OR
PEG NaCl Solution (Swift Biosciences, Cat. No. EC-PEG-48)
Invitrogen DynaMag™, Agencourt® SPRIPlate® or similar magnetic rack for magnetic bead clean-ups
qPCR-based input DNA quantification assay (for FFPE and cfDNA samples)
Qubit or similar fluorometric input DNA quantification assay (for high quality samples such as fresh frozen)
qPCR-based library quantification assay for Illumina libraries
Microcentrifuge
Programmable thermocycler operating within manufacturer’s specifications
0.2 ml PCR tubes or 96-well plate
Aerosol-resistant tips and pipette ranges from 1-1000 µl
200-proof/absolute ethanol (molecular biology grade)
Nuclease-free water (molecular biology grade)
Accel-Amplicon, like any amplicon enrichment technology, poses a risk of contamination of surfaces and other
samples following the amplification step. Please use extreme caution when opening your sample tubes following the
Multiplex PCR step. It is highly recommended that separate workspaces and pipettes be maintained for pre-PCR
and post-PCR steps. A negative pressure hood should be used for post-PCR steps if available. Clean lab areas
using 0.5% sodium hypochlorite (10% bleach) and use specialty barrier pipette tips. Dispose of pipette tips and
other disposables in sealed plastic bags.
www.swiftbiosci.com
5
QUANTIFYING STARTING INPUT MATERIAL
Improper quantification of input material can lead to assay failure. Please read this section carefully and quantify the
types of input material specified below appropriately to ensure success. The limit of detection, sensitivity, and
specificity of Accel-Amplicon Panels is highly dependent on accurate input quantification.
FFPE


Use a qPCR-based assay to quantify starting material with amplicons that are sized to indicate the amplifiable content
of the sample. Accel-Amplicon Panels are designed with amplicons of approximately 140 bp for maximum
compatibility with FFPE DNA. Therefore, using a qPCR assay with amplicons in this size range is recommended.
Please use one of the following options to quantify:
 Commercially available qPCR-based input quantification kit
 A lab-based qPCR test (see Appendix B for published ALU115-qPCR repeat assay)
The following table illustrates how an absorbance-based method (NanoDrop™) and a fluorometric-based method
(Qubit) may overestimate FFPE DNA quantity versus the ALU115-qPCR assay:
FFPE 1
FFPE 2
FFPE 3
FFPE 4
FFPE 5
FFPE 6
FFPE 7
FFPE 8
FFPE 9
FFPE 10
NANODROP
(ng/μl)
7.1
26.2
25.2
35.4
59.9
43.0
67.6
76.6
14.1
246.0
QUBIT
(ng/μl)
2.3
11.4
11.5
15.0
32.4
23.0
35.6
42.2
5.9
84.0
ALU115-qPCR
(ng/μl)
1.5
7.3
10.2
14.7
20.7
16.8
27.4
17.5
3.4
5.8
As shown here, Qubit represents
amplifiable DNA content more accurately
than NanoDrop, however is not as accurate
as the qPCR assay. For sample types with
more consistent high quality DNA including
whole blood, fresh frozen samples, and
cultured cells, quantification by Qubit is a
reliable indicator of amplifiable content.
Circulating, Cell-Free DNA (cfDNA)
 Use a qPCR-based assay to quantify starting material with two differently sized amplicons: (1) a short amplicon to
indicate the amplifiable quantity and (2) a larger amplicon to indicate the molecular weight of the DNA in the sample.
Accel-Amplicon Panels are designed with amplicons of approximately 140 bp for maximum compatibility with cfDNA.
Therefore, using a qPCR assay with amplicons in this size range is recommended. Please use one of the following
options to quantify:
 Commercially available qPCR-based input quantification kit validated for cfDNA assessment
 A lab-based qPCR test with a short amplicon to indicate quantity and a larger amplicon to indicate integrity
 Refer to the technical note, “Assessment of Concentration and Integrity of cfDNA” available within the
Literature content on the Accel-Amplicon product page at www.swiftbiosci.com for ALU115/247 qPCR assay
details and interpretation of cfDNA quantity and purity.
High Quality Genomic DNA, Whole Blood, and Fresh Frozen Tissue
 Use Qubit or a qPCR-based assay to quantify starting material
If you have questions related to FFPE or cfDNA sample quality, please contact Swift technical support at
technicalsupport@swiftbiosci.com or tel: 734.330.2568.
www.swiftbiosci.com
6
PREPARE THE LIBRARY
For best results, please follow these instructions:
 To maximize efficient use of enzyme reagents, remove enzyme tubes from -20 °C storage and place on ice, NOT in a
cryocooler, for at least 10 minutes to allow reagents to reach 4 °C prior to pipetting. Attempting to pipette enzymes
at -20 °C may result in a shortage of enzyme reagents.
 After thawing reagents, briefly vortex all reagents except the enzymes in the Indexing Step (Y2, Y3, Y4) and spin
down in a microcentrifuge.
 Separate the Multiplex PCR Reagents (keep in pre-PCR area) and Indexing Reagents (keep in post-PCR area).
 Prepare reactions on ice before adding to samples and performing incubations.
 Before starting, prepare a fresh 80% ethanol solution using 200-proof/absolute ethanol and nuclease-free water
(approximately 1 ml will be used per sample).
 This “with bead” protocol utilizes a PEG NaCl solution in SPRI Step 2 to bind DNA to SPRIselect beads already in the
tube, rather than adding fresh SPRIselect beads again. To prepare a solution of 20% polyethylene glycol (PEG-8000)
and 2.5 M NaCl:
 Add 10 g of PEG-8000 (Sigma-Aldrich, Cat. No. P5413) and 7.3 g of NaCl to a 50 ml conical tube. Add 20-25 ml
of ultrapure water and mix. When completely dissolved, transfer the solution to a graduated cylinder and adjust
the volume to 50 ml with ultrapure water. Carefully prepare this solution. Improper ratios of PEG and NaCl in this
solution could affect recovery of amplicons and percentage of adapter dimers seen in your sequencing data.
Input Quantification
Quantify the starting material with the appropriate assay (qPCR-based for FFPE and cfDNA, Qubit for high quality DNA
from whole blood, fresh frozen, or cultured cells) as described in the Quantifying Starting Input Material section.
The optimal coverage uniformity, sensitivity, and specificity of this technology are achieved with qPCR-verified input
amounts in the 10-25 ng range. Between 25-100 ng, coverage uniformity may be mildly reduced while sensitivity and
specificity are preserved. Using less than 10 ng may reduce specificity of the assay and affect variant calling for low
frequency alleles. Consider the following example allele frequencies versus sequencing performance:
10 ng
HUMAN
GENOME
EQUIVALENTS
(TOTAL
COPIES)
3000
10 ng
3000
1%
30

1 ng
300
5%
15

1 ng
300
1%
3
SAMPLE
QUANTITY
www.swiftbiosci.com
EXAMPLE
ALLELE
FREQUENCY
EXAMPLE
ALLELE
EQUIVALENTS
(COPIES)
FEASIBILITY OF
CALLING VARIANT
(HIGH QUALITY DNA)
FEASIBILITY OF
CALLING VARIANT
(FFPE)
5%
150


Depends on sample
quality
Depends on sample
quality
Follows Poisson
distribution for
presence of copies
Follows Poisson
distribution for
presence of copies
7
Multiplex PCR Step
1. Load the Multiplex PCR Thermocycler Program (see Appendix A for panel specific program) and allow the block to
reach 98 °C before loading samples (confirm lid heating is turned ON).
2. Load 10 µl of sample DNA (adjust with Pre-PCR TE) into each PCR tube.
3. Make the Multiplex PCR Reaction Mix with the following components. Assemble on ice. Components G1, G2, and G3
should be vortexed first and may be master-mixed when running multiple samples in parallel.
COMPONENT
VOLUME (1 REACTION)
Reagent G1*
Reagent G2
Enzyme G3
Sample DNA
Total
2 μl
3 μl
15 μl
10 µl
30 µl
*Reagent G1 is the Panel-specific set of amplification primers.
4. Mix well and then add 20 µl of the Multiplex PCR Reaction Mix to each 10 µl sample. Place in the thermocycler and run
the program.
Treat PCR products with care to avoid contaminating work areas and other samples. It is highly recommended that
separate workspaces and pipettes be maintained for pre-PCR and post-PCR steps. A negative pressure hood should
be used for post-PCR steps if available. Clean lab areas using 0.5% sodium hypochlorite (10% bleach) and use
specialty barrier pipette tips. Dispose of pipette tips and other disposables in sealed plastic bags.
5. Move samples to post-PCR area before opening tubes. Keep samples at room temperature. At no time should
‘with bead’ samples be stored on ice, as this affects binding to SPRI beads.
6. Make the Indexing Reaction Mix with the following components. Assemble this reaction mix on ice and keep cold
until adding it to samples in the Indexing Step, but leave samples themselves at room temperature in
preparation for SPRI cleanup. All components may be master-mixed when running multiple samples in parallel.
COMPONENT
VOLUME (1 REACTION)
Buffer Y1
Enzyme Y2
Enzyme Y3
Enzyme Y4
Total
31 μl
1 μl
1 μl
2 μl
35 μl
SPRI Step 1
1. Ensure beads and samples are at room temperature. Briefly vortex beads to homogenize before use.
2. Add 36 µl (ratio: 1.2) of SPRIselect beads to each 30 µl sample. Mix by vortexing. Ensure no bead-sample suspension
droplets are left on the sides of the tube.
3. Incubate the samples for 5 minutes at room temperature off the magnet.
4. Pulse-spin the samples in a microfuge. Place the sample tubes on a magnetic rack until the solution clears and a pellet
is formed (≈ 5 minutes).
5. While leaving your sample on the magnet, remove and discard the supernatant without disturbing the pellet
(approximately 5 µl may be left behind). Leave tubes on the magnet.
6. Add 200 μl of freshly prepared ethanol solution to the pellet while it is still on the magnet. Use care not to disturb the
pellet. Incubate for 30 seconds, and then carefully remove the ethanol solution.
7. Repeat step 6 once for a second wash with the ethanol solution.
8. Pulse-spin the samples in a microfuge, place back onto the magnet and remove any residual ethanol solution from the
bottom of the tube.
9. Air-dry the pellet briefly, watching the pellet to avoid cracking or over-drying. Leave tubes on the magnet. Proceed to
the Indexing Step for resuspension without delay.
www.swiftbiosci.com
8
Indexing Step
1.
2.
3.
4.
5.
Continue working in the post-PCR area.
Load the Indexing Thermocycler Program and allow the block to reach 37 °C before loading samples.
Add a unique combination of 5 µl Index D50X + 10 µl Index D7XX to each sample bead pellet.
Add 35 µl of the cold Indexing Reaction Mix to each sample and resuspend the pellet (total volume 50 µl).
Place in the thermocycler and run the program (37 °C for 20 minutes).
SPRI Step 2
1. Ensure PEG NaCl solution is at room temperature. Briefly vortex the PEG NaCl solution to homogenize before use.
2. Add 42.5 µl (ratio: 0.85) of PEG NaCl solution to each 50 µl sample. Mix by vortexing. Ensure no bead-sample
suspension droplets are left on the sides of the tube.
3. Incubate the samples for 5 minutes at room temperature off the magnet.
4. Pulse-spin the samples in a microfuge. Place the sample tubes on a magnetic rack until the solution clears and a pellet
is formed (≈ 5 minutes).
5. While leaving your sample on the magnet, remove and discard the supernatant without disturbing the pellet
(approximately 5 µl may be left behind). Leave tubes on the magnet.
6. Add 200 μl of freshly prepared ethanol solution to the pellet while it is still on the magnet. Use care not to disturb the
pellet. Incubate for 30 seconds, and then carefully remove the ethanol solution.
7. Repeat step 6 once for a second wash with the ethanol solution.
8. Pulse-spin the samples in a microfuge, place back onto the magnet and remove any residual ethanol solution from the
bottom of the tube.
9. Air-dry the pellet briefly, watching the pellet to avoid cracking or over-drying. Leave tubes on the magnet.
10. Add 20 µl of Post-PCR TE buffer and resuspend the pellet, mixing well by pipetting up and down until homogenous.
Incubate at room temperature for 2 minutes off the magnet. Then place the sample back on the magnet and transfer
the clean 20 µl library eluate to a fresh tube. Ensure that eluate does not contain magnetic beads (indicated by brown
coloration in eluate). If magnetic beads are present, pipette eluate into a new tube, place on magnet, and transfer
eluate again.
Library Quantification
Quantify a 1:100,000 dilution of your library in triplicate using a qPCR assay based upon a library size of 265 bp.
Improper library quantification by other methods will lead to uneven pooling and suboptimal cluster density,
impacting sequencing data.
It is not recommended to use a Bioanalyzer for quantifying libraries because:
 As there is no PCR enrichment of the library following the Indexing Step, the Bioanalyzer will not accurately quantify
fully adapted library vs. other DNA.
 Library adapters have secondary structure which exhibits migration artifacts on the Bioanalyzer.
It is not recommended to use a fluorometric method (such as Qubit) for quantifying libraries because:
 As there is no PCR enrichment of the library following the Indexing Step, a fluorometric method will not accurately
quantify fully adapted library vs. other DNA.
Bioinformatics Options
As noted in the Appendix B section of this manual titled Sample Sheet – Special Considerations, please ensure that
adapter trimming is enabled while setting up the sequencing run.
A primer trimming technical note and FASTA files are provided with purchase of the Accel-Amplicon Panel. If additional
informatics pipeline advice is needed to use these instructions, please contact Swift technical support at
technicalsupport@swiftbiosci.com or tel: 734.330.2568.
www.swiftbiosci.com
9
APPENDIX A: PANEL-SPECIFIC INFORMATION
56G Oncology Panel
Cat. No. AL-IL56G-12/48
This panel contains 263 amplicons sized 92-184 bp (average 138 bp) that cover hotspots and contiguous regions of 56
genes with a total target size of 23.7 kbp.

Pre-program a thermocycler with the following programs to expedite the workflow:
Lid heating ON
Multiplex PCR
Thermocycler Program
Indexing
Thermocycler Program
30 sec
98 °C
10 sec
98 °C
5 min
63 °C
1 min
65 °C
10 sec
98 °C
1 min
64 °C
1 min
65 °C
Hold
4 °C
Lid heating OFF
20 min
4 cycles
21 cycles
37 °C
NOTE: The number of amplification cycles is precisely optimized to produce the best results and is based on qPCRverified input quantity.

MiSeq Multiplexing guidelines are as follows:
MiSEQ REAGENT KIT
v2 Nano (300 cycles)
(2M paired-end reads)
v2 Micro (300 cycles)
(8M paired-end reads)
v2 (300 cycles)
(30M paired-end reads)
v3 (600 cycles)
(50M paired-end reads)
INTENDED READ DEPTH
LEVEL OF MULTIPLEXING
1000X
2500X
5000X
1000X
2500X
5000X
1000X
2500X
5000X
1000X
2500X
5000X
7
3
1
30
12
6
114*
45
22
190*
76
38
*Requires custom indexing solution. Please inquire.
www.swiftbiosci.com
10
Comprehensive TP53 Panel
Cat. No. AL-ILTP53-12/48
This panel contains 21 amplicons sized 106-154 bp (average 140 bp) that comprehensively cover all coding regions of
TP53 with a total target size of 1.8 kbp.

Pre-program a thermocycler with the following programs to expedite the workflow:
Multiplex PCR
Thermocycler Program
Indexing
Thermocycler Program
Lid heating ON
30 sec
98 °C
10 sec
98 °C
5 min
63 °C
1 min
65 °C
10 sec
98 °C
1 min
64 °C
1 min
65 °C
Hold
4 °C
Lid heating OFF
20 min
4 cycles
22 cycles
37 °C
NOTE: The number of amplification cycles is precisely optimized to produce the best results and is based on qPCRverified input quantity.

MiSeq Multiplexing guidelines are as follows:
MiSEQ REAGENT KIT
v2 Nano (300 cycles)
(2M paired-end reads)
v2 Micro (300 cycles)
(8M paired-end reads)
v2 (300 cycles)
(30M paired-end reads)
v3 (600 cycles)
(50M paired-end reads)
INTENDED READ DEPTH
LEVEL OF MULTIPLEXING
1000X
2500X
5000X
1000X
2500X
5000X
1000X
2500X
5000X
1000X
2500X
5000X
95
38
19
192*
152*
76
192*
192*
192*
192*
192*
192*
*Requires custom indexing solution. Please inquire.
www.swiftbiosci.com
11
EGFR Pathway Panel
Cat. No. AL-ILEGFR-12/48
This panel contains 17 amplicons sized 107-155 bp (average 136 bp) that cover hotspots in BRAF, KRAS, and NRAS,
and contiguous regions of EGFR, with a total target size of 1.5 kbp.

Pre-program a thermocycler with the following programs to expedite the workflow:
Multiplex PCR
Thermocycler Program
Indexing
Thermocycler Program
Lid heating ON
30 sec
98 °C
10 sec
98 °C
5 min
63 °C
1 min
65 °C
10 sec
98 °C
1 min
64 °C
1 min
65 °C
Hold
4 °C
Lid heating OFF
20 min
4 cycles
23 cycles
37 °C
NOTE: The number of amplification cycles is precisely optimized to produce the best results and is based on qPCRverified input quantity.

MiSeq Multiplexing guidelines are as follows:
MiSEQ REAGENT KIT
v2 Nano (300 cycles)
(2M paired-end reads)
v2 Micro (300 cycles)
(8M paired-end reads)
v2 (300 cycles)
(30M paired-end reads)
v3 (600 cycles)
(50M paired-end reads)
INTENDED READ DEPTH
LEVEL OF MULTIPLEXING
1000X
2500X
5000X
1000X
2500X
5000X
1000X
2500X
5000X
1000X
2500X
5000X
110
44
22
192*
184*
92
192*
192*
192*
192*
192*
192*
*Requires custom indexing solution. Please inquire.
www.swiftbiosci.com
12
Sample_ID Panel
Cat. No. AL-ILSID-48
This panel contains 104 amplicons sized 120-160 bp (average 145 bp) that cover exonic SNPs with high minor allele
frequency and gender identification targets.

Pre-program a thermocycler with the following programs to expedite the workflow:
Multiplex PCR
Thermocycler Program
Indexing
Thermocycler Program
Lid heating ON
30 sec
98 °C
10 sec
98 °C
5 min
63 °C
1 min
65 °C
10 sec
98 °C
1 min
64 °C
1 min
65 °C
Hold
4 °C
Lid heating OFF
20 min
4 cycles
22 cycles
37 °C
NOTE: The number of amplification cycles is precisely optimized to produce the best results and is based on qPCRverified input quantity.


In order to use the information from the Sample_ID Panel to properly discriminate between samples, it is
recommended to sequence the panel to a minimum depth of 200-500X. This corresponds to between 34-90 samples
multiplexed on a MiSeq v2 Nano (300 cycles, 2M paired-end reads).
Compare the target profile across samples of interest to confirm a match. Because each target will exhibit a presence
level in each sample of none (~0%), heterozygous (~50%), or homozygous (~100%), examining all 104 targets in
combination with gender identification will reveal a unique profile for the sample that can be compared to other
samples. For additional information about the targets utilized and their interpretation, please consult the following
publications:
 Pengelly RJ, Gibson J, Andreoletti G, Collins A, Mattocks CJ, Ennis S. A SNP profiling panel for sample tracking
in whole-exome sequencing studies. Genome Med. 2013 Sep 27;5(9):89.
 Butler E, Li R. Genetic Markers for Sex Identification in Forensic DNA Analysis. J Forensic Investigation.
2014;2(3): 10.
www.swiftbiosci.com
13
APPENDIX B: ACCEL-AMPLICON SUPPORTING
INFORMATION
ALU115-qPCR Input Quantification Assay



Highly abundant ALU sequences can be used for the sensitive quantification of input DNA, as presented by T.B. Hao
in the British Journal of Cancer (2014) 111, 1482–1489 for quantifying cfDNA. The following primer sequences were
used to amplify short (115 bp; ALU115) amplicons from genomic ALU repeats. ALU115-qPCR results accurately
detect the total quantity of DNA.
 ALU115 (forward) 5’-CCTGAGGTCAGGAGTTCGAG-3’
 ALU115 (reverse) 5’-CCCGAGTAGCTGGGATTACA-3’
Reactions of 20 μl were assembled for each ALU-qPCR and contained a final concentration of 200 nM of each primer,
1X of iTaq™ Universal SYBR® Green Supermix (Bio-Rad, Catalog# 172-5120) and the appropriate amount of
standard DNA or cfDNA. A standard curve was generated from serial dilutions (22ng, 2.2ng, 0.22ng, 0.022ng,
0.0022ng) of human genomic DNA from Promega (Catalog# G3041) and a no template control. The reaction was
conducted on a real-time thermocycler (Bio-Rad CFX96) as follows:
 95 °C for 3 minutes
 30 cycles: 95 °C for 5 seconds, 62 °C for 30 seconds, Read SYBR
A melting curve can also be performed to ensure that only one peak was amplified for all samples.
Sample Sheet – Special Considerations




Open Illumina Experiment Manager and create a sample sheet.
On the Instrument selection page, select “MiSeq”.
In the MiSeq Application Selection page, select category “Other” and select application “FASTQ Only”.
On the workflow parameter page:
 Enter the Reagent Cartridge barcode.
 Select “TruSeq HT” as the Sample Prep Kit.
 Index Reads: “2”.
 Read Type: “Paired End”.
 Cycles Read 1: “151”, Cycles Read 2: “151”.
 Make sure the “Use Adapter Trimming” and “Use Adapter Trimming Read 2” are selected.
MiSeq Loading Recommendations
Load the MiSeq between 10-12 pM. These recommendations are for the MiSeq v2 Reagent Kits.
Amplicon libraries can be pooled together to obtain a 2 nM or 4 nM final concentration mix. Denaturation of libraries with
freshly diluted 0.2 N NaOH is required before loading on the MiSeq.


2 nM library denaturation (supports 10 pM loading)
 2 nM library pool (5 µl) + 0.2N NaOH (5 µl).
 Mix and incubate 5 minutes at room temperature.
 Add 990 µl of pre-chilled HT1 to obtain a 10 pM denatured library mix, mix well.
 Load 600 µl in the cartridge.
4 nM library denaturation (supports 10 pM-20 pM loading)
 4 nM library pool (5 µl) + 0.2N NaOH (5 µl).
 Mix and incubate 5 minutes at room temperature.
 Add 990 µl of pre-chilled HT1 to obtain a 20 pM denatured library pool, mix well.
 Dilute the denatured DNA to load the cartridge:
FINAL CONCENTRATION
10 PM
11 PM
12 PM
20 pM Denatured Libraries
Pre-chilled HT1
300 µl
300 µl
330 µl
270 µl
360 µl
240 µl

Load 600 µl of the desired dilution into the cartridge.
www.swiftbiosci.com
14
Troubleshooting Common Problems
PROBLEM
POSSIBLE CAUSE
SUGGESTED REMEDY
Lower than expected yields.
Inadequate sample quality and/or
quantity, incorrect input quantification
method, or incorrect SPRI methods.
Incomplete resuspension of beads
after ethanol wash during SPRI steps.
Lower than expected cluster density.
Over-drying of beads.
Use 25 ng of qPCR-quantified input
and extend the incubation time for the
Indexing Step from 20 minutes to 60
minutes. Perform SPRI carefully.
Continue pipetting the liquid over the
beads for complete resuspension.
Quantify library with a qPCR-based
method for flow cell loading
calculations.
Unusual Bioanalyzer trace.
Error in library quantification.
Bioanalyzer and Qubit do not
accurately quantify fully adapted
library vs. other DNA.
Secondary structure of adapters and
lack of PCR enrichment of the library
following the Indexing Step causes
migration artifacts on Bioanalyzer.
Quantify library with a qPCR-based
method; ascertain amplicon insert size
from the sequencing data.
If you experience problems with your library prep, please contact us at technicalsupport@swiftbiosci.com, or by
phone: 734.330.2568 (9:00 am – 5:00 pm ET, Monday through Friday).
www.swiftbiosci.com
15
Indexed Adapter Sequences
During the Indexing Step in the protocol, you must use a unique combination of Index Adapters to re-suspend and label
each library. Libraries made with uniquely indexed adapter combinations may be multiplexed during cluster generation
and co-sequenced on the same Illumina flow cell.
CONTENTS: Unique indexed adapters, which should be used where this manual calls for 5 or 10 μl of each Index Primer:
D5 ADAPTERS
SEQUENCE
D501
D502
D503
D504
D505
D506
D507
D508
TATAGCCT
ATAGAGGC
CCTATCCT
GGCTCTGA
AGGCGAAG
TAATCTTA
CAGGACGT
GTACTGAC
D7 ADAPTERS
SEQUENCE
D701
D702
D703
D704
D705
D706
D707
D708
D709
D710
D711
D712
ATTACTCG
TCCGGAGA
CGCTCATT
GAGATTCC
ATTCAGAA
GAATTCGT
CTGAAGCT
TAATGCGC
CGGCTATG
TCCGCGAA
TCTCGCGC
AGCGATAG
The number on the product tube label indicates which indexed adapter is provided in the tube. During library prep, make
sure to note which indexed adapter combination you are using with your sample and do not use the same indexed
adapter combination on two different samples you plan to co-sequence.
www.swiftbiosci.com
16
General Warranty
Swift Biosciences, Inc. (“Swift”) warrants that its products meet Swift’s specifications at the time of delivery. Any sample
or model used in connection with Swift's product literature is for illustrative purposes only and does not constitute a
warranty that the products will conform to the sample or model.
To the maximum extent permitted by applicable law, Swift hereby expressly disclaims, and the buyer hereby expressly
waives, any warranty regarding results obtained through the use of the products including, without limitation, any claim of
inaccurate, invalid, or incomplete results. All other warranties, representations, terms and conditions (statutory, express,
implied or otherwise) as to quality, condition, description, merchantability, fitness for purpose, or non-infringement (except
for the implied warranty of title) are hereby expressly excluded.
All warranty claims on products must be made in writing within ninety (90) days of receipt of the products. Swift’s sole
liability and the buyer’s exclusive remedy for a breach of this warranty is limited to replacement or refund at the sole
option of Swift.
The warranties identified in this paragraph are Swift's sole and exclusive warranties with respect to the products
and are in lieu of all other warranties, statutory, express or implied, all of which other warranties are expressly
disclaimed, including without limitation any implied warranty of merchantability, fitness for a particular purpose,
non-infringement, or regarding results obtained through the use of any product (including, without limitation, any
claim of inaccurate, invalid or incomplete results), whether arising from a statute or otherwise in law or from a
course of performance, dealing or usage of trade.
Limitation of Liability
Swift Biosciences, Inc. (“Swift”) shall have no liability under the warranties cited above with respect to any defect in the
products arising from: (i) specifications or materials supplied by the buyer; (ii) wilful damage or negligence of the buyer or
its employees or agents; (iii) abnormal working conditions at the buyer's premises; (iv) failure to follow Swift's use restrictions
or instructions (whether oral or in writing); (v) misuse or alteration of the products without Swift's approval; or (vi) if the buyer
is in breach of its payment obligations in regards to purchasing the products.
To the fullest extent allowed by law, in no event shall Swift be liable, whether in contract, tort, strict liability,
negligence, warranty, or under any statute or on any other basis for any special, incidental, indirect, exemplary,
punitive, multiple or consequential damages sustained by the buyer or any other person or entity arising out of
or caused by product, Swift's performance or failure to perform its obligations relating to the purchase of product
or performance of services, Swift's breach of these terms, the possession or use of any product, or the
performance by Swift of any services, whether or not foreseeable and whether or not Swift is advised of the
possibility of such damages, including without limitation damages arising from or related to loss of use, loss of
data, downtime, procurement of substitute products or services, or for loss of revenue, profits, goodwill, or
business or other financial loss.
The total liability of Swift arising under or in connection with the purchase of the products, including for any breach of
contractual obligations and/or any misrepresentation, misstatement or tortious act or omission (including without limitation,
negligence and liability for infringement of any third party intellectual property rights) shall be limited to damages in an
amount equal to the amount paid to Swift under the purchase agreement.
The exclusion of liability shall apply only to the extent not prohibited by applicable law.
Notice to Purchaser: Limited License
This product is for research use only and is licensed to the user under Swift Biosciences intellectual property only for the
purchaser’s internal purposes. Not for use in diagnostic procedures.
www.swiftbiosci.com
17
NOTES
Swift Biosciences, Inc.
58 Parkland Plaza, Suite 100 • Ann Arbor, MI 48103 • 734.330.2568 • www.swiftbiosci.com
© 2016, Swift Biosciences, Inc. The Swift logo and Accel-Amplicon are trademarks of Swift Biosciences. This product is for Research Use Only. Not for use in diagnostic
procedures. Illumina is a registered trademark of Illumina, Inc. Agencourt is a registered trademark and SPRIselect and SPRIPlate are trademarks of Beckman Coulter,
Inc. NanoDrop, Qubit, and SYBR are registered trademarks and DynaMag is a trademark of Thermo Fisher Scientific, Inc. iTaq is a trademark of Bio-Rad Laboratories,
Inc. Oligonucleotide sequences © 2016 Illumina, Inc. All rights reserved. 16-0738, 03/16