Shoreline Complete for Illumina Technical Manual
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Shoreline Complete™ for Illumina Technical Manual
Contents Introduction to Shoreline Complete™ Kits................................................................................................ 3 Kit Components ....................................................................................................................................... 3 Important Parameters ............................................................................................................................. 3 Storage buffer...................................................................................................................................... 3 Sample Types and Input Quantities ...................................................................................................... 3 DNA Quantitation ................................................................................................................................ 4 Target Reads per Sample ..................................................................................................................... 4 Multiplexing Barcodes ......................................................................................................................... 4 Amplicon Normalization ...................................................................................................................... 4 Microbiome Controls ........................................................................................................................... 5 Safe Stopping Points ............................................................................................................................ 5 Sample Storage .................................................................................................................................... 5 Equipment and reagents supplied by user............................................................................................ 5 Protocols ................................................................................................................................................. 6 Shoreline Complete™ Lyse Protocol ..................................................................................................... 6 Shoreline Complete™ Purify Protocols ................................................................................................. 8 Shoreline Complete™ Amplify Protocol ................................................................................................ 9 Amplicon QC .......................................................................................................................................... 10 Post Amplification Clean-up ................................................................................................................... 10 Pooled Amplicon QC .............................................................................................................................. 11 Sequencing ............................................................................................................................................ 12 References............................................................................................................................................. 12 Appendix A: Barcode Sequences for V1-V3 and V4 Set A ........................................................................ 13 Appendix B: Barcode Sequences for V1-V3 and V4 Set B ........................................................................ 16 Appendix C: Barcode Sequences for V4 Set C ......................................................................................... 19
Introduction to Shoreline Complete™ Kits Shoreline Complete™ kits are a complete solution for bacterial 16S microbiome profiling studies, providing investigators with materials and protocols for rapid DNA preparation and Illumina sequencing library generation. Each kit contains reagents for comprehensive bacterial lysis, extraction, and purification of DNA, individual sample barcoding for up to 96 samples, and amplification of variable regions of the 16S gene for microbiome analysis. DNA can be extracted from a wide variety of samples including fecal, swab, tissue, and saliva. DNA is amplified using dual-unique barcoded primers targeting the V1-V3 or V4 variable region of the 16S rRNA gene. The single-step PCR, made up of primers containing the Illumina adapter sequence, dual- unique barcodes, and a diverse set of target specific primers covering known variations in the primer sites , generates ready-to-pool amplicon libraries to be sequenced on the Illumina platform. This document provides guidance for using the Shoreline Complete™ kits for metagenomic studies sequenced on the Illumina platform. Kit Components Kit Contents for Shoreline Complete™ Lyse and Purify 1. Lysis-1, dried solution in plate 2. Purification Buffer Kit Contents for Shoreline Complete™ Amplify 1. Barcoded primers, dried in plate 2. 2X PCR Premix 3. PCR sealing film Important Parameters Storage buffer Most sample storage buffers are compatible with the lysis, however for buffers with pH < 5 we recommend adding 10 µL sample, to prevent neutralization of the Shoreline Complete™ lysis buffer. Sample Types and Input Quantities Shoreline Complete™ kits have been validated with the sample types specified in Table 1, below. For Sample Type of: Use: Solid fecal Liquid fecal stored in other buffers Protocol for DNA Genotek OMNIgene®•GUT frozen/liquid/mouse fecal, OMNIgene® Gut Tissue Samples Mouse fecal pellet Swabs Protocol for skin, swabs, Saliva cell pellets, saliva Pelleted cells Table 1. Validated Sample Types
Table 2 (below) provides the recommended input quantities for each of the sample types. Please note that adding less than the recommended amount may not yield sufficient amounts of DNA, while using more than the maximum quantity may cause insufficient lysis. Sample Type Recommended Amount Maximum Amount Solid fecal 1-3 mg 10 mg Liquid fecal 10 µL 10 µL DNA Genotek OMNIgene®•GUT 10 µL 10 µL Tissue 20 mg 30 mg Mouse fecal 5 mg (1/4 pellet) 10 mg (1/2 pellet) Bacterial Cells 106 cells 108 cells Table 2. Sample Type Input Quantities DNA Quantitation The Shoreline Complete PCR has been optimized to handle a broad range of input DNA, therefore, it is not necessary to quantify DNA prior to amplification. However, if quantitation is desired, keep in mind that the Shoreline Complete™ lysis protocol results in single stranded DNA. We recommend the Qubit™ ssDNA Quantification kit. DNA quantification by NanoDrop™ is not recommended. Target Reads per Sample The required reads per sample are dependent on goals of each sequencing project. General guidelines recommend 100,000 reads per sample, but under some circumstances, sufficient taxonomic classification can be obtained using fewer reads, a tradeoff allowing for a higher level of sample multiplexing. It is not recommended to pool different size amplicons (V1-V3 and V4) into a single sequencing run. Multiplexing Barcodes The Shoreline Complete™ kits contain primers with dual-unique indexing. This barcoding approach is an improvement over the typical combinatorial approach due to reduced index hopping.1 For V4 kits, 288 barcodes are available and for V1-V3, 192 barcodes are available. See the Table 3 and Table 4 below for permissible barcode combinations. See Appendix A, Appendix B, and Appendix C for barcode sequences for Set A, Set B, and Set C, respectively. V1-V3 V4 Set A, Set B Set A, Set B, Set C Table 3. V1-V3 barcode sets Table 4. V4 barcode sets Amplicon Normalization Using an equi-volume pooling approach for unpurified PCR products is the quickest and most convenient way to normalize amplicon libraries. Equi-volume pooling is rapid and generally provides good representation of samples when used in combination with visualization on a gel,
Bioanalyzer, or equivalent. Other normalization methods are acceptable, including equimolar pooling based on fluorometry or qPCR, however, these methods may be more expensive or labor intensive. Equimolar pooling may be beneficial if processing multiple sample types and/or samples that are expected to have widely varying microbial loads2. Microbiome Controls Several controls are recommended to monitor the workflow including lysis and amplification efficiency, as well as environmental contamination. Recommended controls and control materials are provided in Table 5. Control Type Recommended Control Material Process Positive Mock community DNA (commercially available) Process Negative Water at the Lysis step PCR Positive Mock community DNA (commercially available) PCR Negative Water Table 5. Recommended controls and control materials Safe Stopping Points Safe stopping points, where the process can be paused, are marked with this symbol. Follow the storage recommendations. Sample Storage Genomic DNA can be stored at +4°C for up to 10 days, or frozen at -20°C for 3 months. Avoid repeated freeze/thaws. Amplicons and library can be stored at 4°C for up to 1 week or -20°C for up to 1 month. Equipment and reagents supplied by user Additional Items Required for Lyse and Purify ● 96-well PCR plate ● Plate sealing film ● Sterile Laboratory Grade Water ● 70% ethanol ● TE Buffer - 10mM Tris, 1mM EDTA, pH 8.0 ● 0.4M KOH Solution ● Magnetic Rack (Invitrogen Dyna Mag 96 side magnetic rack, #12331D or Magnetic stand-96 #AM10027) Additional Items Required for Amplify ● Qiagen MinElute® PCR Purification Kit (Cat. No. 28004) ● Sterile Laboratory Grade Water ● Sterile 100% Ethanol Suggested items for rapid processing
● Repeater Pipette with multi-volume tips ● Single Channel Pipettes ● Multichannel Pipette ● Sterile inoculating loops Suggested items for sample QC ● 0.8% agarose gel with ethidium bromide or other dsDNA stain ● Gel loading dye ● 2kb DNA ladder ● Bioanalyzer reagents or equivalent Protocols Shoreline Complete™ Lyse Protocol 1. Use the following calculation to make 0.4 M potassium hydroxide (KOH) solution. Make solution fresh daily. × 44.56 = 0.4 ℎ : = , = , 2. Make 70% Ethanol. Add 17.5 mL 100% ethanol to 7.5 mL sterile laboratory grade water 3. Bring Purification Buffer (white capped bottle) to room temperature. 4. Remove film from the 96-well PCR plate containing dried Lysis-1. 5. Follow the instructions for the specific Sample Type in Table 6, below: CAUTION: Avoid over-mixing to prevent foaming of the Lysis-1 reagent. Sample Type Sample type-specific Lysis Instructions Add 50 µL Molecular Biology Grade water to each well in the 96-well Lysis-1 plate. Using sterile inoculating loop, transfer ~3 mg of each fecal sample to the corresponding well (Figure 1 and Figure 2). Spin each loop to disperse the sample in Lysis-1, and discard loop. Frozen Solid Fecal Samples Figure 1. Figure 2. Add 50 µL Molecular Biology Grade water to each well in the 96-well Lysis-1 plate. Mouse Fecal Pellet Place up to 5 mg (1/4 mouse pellet) in each well. DNA Genotek Add 40 µL Molecular Biology Grade water to each well in the 96-well Lysis-1 plate. OMNIgene®•GUT Sample Add 10 µL of sample from the OMNIgene®•GUT Sample Collection Tube to Lysis-1. Collection Tube
Liquid Fecal Samples stored Add 40 µL Molecular Biology Grade water to each well in the 96-well Lysis-1 plate. in other buffers Add 10 µL of sample. Add 50 µL Molecular Biology Grade water to each well in the 96-well Lysis-1 plate. Tissue Samples Place 20mg tissue sample in each well. Centrifuge saliva samples at 5500 RCF for 10 minutes to pellet cells. Remove and discard supernatant. Saliva Re-suspend in 50 µL of Molecular Biology Grade water, TE, or PBS. Transfer 50 µL of cell suspension to Lysis-1 well. Moisten swab with buffer containing Tris-EDTA and 0.5% Tween 20. Collect sample from skin. Place swab in 1.7 mL microcentrifuge tube. Break the swab handle off at designated location. Centrifuge tube with swab for 5 minutes at 10000 RCF. Skin Swab Remove swab and transfer supernatant to a clean 1.7 mL tube. Centrifuge tube with swab second time for 5 minutes at 10000 RCF. Remove swab and combine supernatant with the first supernatant. Discard spent tube and swab. Transfer 50 µL of the supernatant to Lysis-1 well. Add 50 µL water, TE, or PBS to cells. Cell Pellet Pipette to mix. Transfer 50 µL of cell suspension to Lysis-1 well. Table 6. Sample type-specific instructions for lysis 6. Add 50 µL 0.4M KOH to each well position in the 96-well plate. Mix briefly by pipetting up and down. Do not over-mix to avoid foaming. A precipitate will form in the wells. 7. Cover the sample plate with plate film, load the sample plate into the thermocycler, close and lock the lid, and heat samples to 95°C for 5 minutes. Precipitate will dissolve.
Shoreline Complete™ Purify Protocols Follow the purification protocol from Table 7 below based on the sample type being used. Protocol for frozen/liquid/mouse fecal, Protocol for skin, swabs, cell pellets, OMNIgene® Gut saliva ● Remove the plate from the ● Remove the plate from the thermocycler thermocycler. ● Place sample plate on ice for 2 ● Vortex two bottles of Purification Buffer minutes. (Precipitate will re-form) (white cap) to re-suspend any brown ● Vortex Purification Buffer bottle magnetic beads that may have settled. (white cap) to re-suspend any brown ● Add 100 µL of Purification Buffer magnetic beads that may have settled. directly to each well of the sample plate ● Add 50 µL of Purification Buffer to and pipette to mix. each well of a new plate for sample ● Cover the sample plate with a new plate purification film. ● After incubating sample plate on ice, spin sample plate in centrifuge at 2000 RCF for 3 minutes (or 400 RCF for 7 minutes) to pellet precipitate to bottom of wells (see Figure 3 below) ● Carefully transfer 50 µL of supernatant into the appropriate well of the clean 96-well plate with Purification Buffer prepared as per the instructions above; and pipette to mix. ● Cover the sample plate with a new plate film. Table 7. Purification protocols based on sample type Figure 3. Pelleted precipitate in bottom of wells after spin 1. Load sample plate onto the thermocycler, close and lock the lid, and incubate solution for 5 minutes at 50˚C to allow DNA to bind to beads 2. After incubation, place plate on magnetic rack to pellet beads with bound DNA (~60 seconds), carefully remove plate film. 3. Once all beads are pelleted, remove all supernatant SLOWLY and discard. Avoid aspirating beads. 4. Leave the plate on the magnet and add 200 µL 70% ethanol to each well. After 30 seconds, remove the supernatant.
5. Repeat the wash from step 4 above. 6. Remove the ethanol completely and let air dry for 2 - 3 minutes. Over-drying will make eluting DNA more difficult, so watch to ensure ethanol is mostly gone but bead pellet does not begin to shrink and crack. 7. Remove plate from magnet. 8. Add 20 µL of 1X TE buffer to each well and briefly pipette up and down to re-suspend beads. 9. Incubate plate at room temperature for 2 minutes. 10. Place plate on magnetic rack for 30 - 60 seconds to pellet magnetic beads. DNA is now in solution. 11. Transfer supernatant containing eluted DNA into a clean plate on ice. 12. Remove plate with pelleted beads from rack. 13. Add another 20 µL 1X TE buffer onto bead pellet, gently pipette up and down to re-suspend beads. 14. Incubate plate for 2 minutes at room temperature. 15. Return plate to magnetic rack. Allow beads to pellet to sides for 30 - 60 seconds. 16. Remove supernatant containing DNA and combine with first eluate in clean plate, avoiding pelleted beads. 17. Dilution: 17.1. For all fecal samples: Dilute 1:5 by adding 160 µl 1X TE buffer to 40 µl of combined eluted DNA for a total of 200 µl. 17.2. For all other samples: Do not dilute. 18. Proceed to Amplify step or store eluted DNA at +4°C. Genomic DNA can be stored at +4°C for up to 10 days, or frozen at -20°C for 3 months. Shoreline Complete™ Amplify Protocol 1. Remove the plate seal from the Amplicon Primer plate. 2. Add 10 µL 2X PCR Premix (blue cap) to each position in the 96-well plate. 3. Add 10 µL Shoreline Biome DNA to each of the 96-wells. 4. Using the PCR sealing film provided with the kit, cover the plate and seal tightly with no gaps. 5. Primers will dissolve off the bottom of each well. Observe that blue color is uniformly distributed throughout 20 µL reaction, if not, tap plate gently until uniform blue color is achieved in each well. 6. Spin plate briefly if reaction mix is not completely at the bottom of the wells. 7. Transfer the plate to thermocycler; close and lock the lid. 8. Run the appropriate PCR protocol for the Shoreline Complete™ kit from Table 8 or Table 9 below.
V1-V3 V4 Heated lid @ 100°C Heated lid @ 100°C Temperature Time Ramp Speed Cycles Temperature Time Ramp Speed Cycles 95°C 3 min 1 cycle 95°C 3 min 1 cycle 95°C 10 sec 95°C 20 sec 34 34 57°C 10 sec 0.5°C/sec 58°C 15 sec 4°C/sec cycles cycles 72°C 21 sec 72°C 15 sec 73°C 45 sec 1 cycle 72°C 2 min 1 cycle 4°C hold hold 4°C hold hold Table 8. PCR Protocol for V1-V3 Kits Table 9. PCR Protocol for V4 Kits Amplicons can be stored at 4°C for up to one (1) week or -20°C for up to one (1) month Amplicon QC Check individual reactions on gel: Run 1.5 µL of sample with 5 µL diluted gel loading dye on 0.8% agarose gel in TBE, 150V for approximately 45 minutes, with DNA ladder. Band should be present at ~650bp V1-V3 and ~450bp for V4. Agilent Bioanalyzer or equivalent can also be used. Post Amplification Clean-up Qiagen MinElute® PCR Purification Protocol 1. Follow MinElute instructions for adding Ethanol (96% - 100%) to PE buffer, and label tubes. 2. Pool 5 µL of each amplified sample from the 96-well plate (when pooling multiple plates, pool 5 µL of each amplified sample from all wells of all plates) into a clean 1.5 mL microcentrifuge tube. (If the amplicon QC gel shows variation in amount of PCR product, volume adjustments can be made here, during pooling.) Save 2 µL of pooled unpurified DNA for gel comparison with purified DNA in the last step below. 3. Combine 80 µL of pooled sample with 400 µL of Qiagen PB buffer. 4. Transfer all of the resulting solution into the MinElute column with provided 2 mL collection tube. 5. Centrifuge the MinElute column at 17,900 RCF for 1 minute or until solution has passed through the column. Discard the flow-through in the collection tube and return the column into the empty tube. 6. Add 750 µL Buffer PE to the MinElute column and centrifuge at 17,900 RCF for 1 minute; discard flow through and return MinElute column to the collection tube. Centrifuge for 1 minute at 17,900 RCF to remove residual Ethanol. 7. Place MinElute column in a clean 1.5 mL microcentrifuge tube. 8. Add 50 µL EB Buffer (10mM Tris-Cl, pH 8.5) or water directly onto center of the MinElute membrane. Incubate column for 1 minute at RT, and then centrifuge column at 17,900 RCF for 1 minute.
Safe stopping point - Pooled undiluted library can be stored at 4°C for up to one (1) week or -20°C for up to one (1) month 9. Quantify the library by a fluorometric method (e.g. Qubit). Determine the size distribution by running the pooled amplicon library on Agilent Bioanalyzer or equivalent. (see Pooled Amplicon QC, below). 10. Use the following formula to convert from ng/μl to nM: × 106 = (660 ) × ℎ : = , = = , 11. Dilute the library to 4nM using the above equation and proceed to the Illumina MiSeq System Denature and Dilute Libraries Guide. Follow the procedure for Protocol A: Standard Normalization Method and MiSeq Reagent Kit v3 (see Sequencing section below). Pooled Amplicon QC Below is an example of the library traces for V4 (Figure 4) and V1-V3 (Figure 5 and Figure 6). Note that depending on composition of the bacterial population, V1-V3 traces can result in single or bimodal peaks3 Figure 4. V4 sample trace
Figure 5. V1-V3 sample trace B Figure 6. V1-V3 sample trace C Sequencing High resolution taxonomic classification is dependent on sequencing quality. Shoreline Complete™ kits should be run on Illumina MiSeq instrument using v3 600-cycle chemistry. This enables the user to perform paired-end 300bp reads, resulting in higher quality reads and allowing better taxonomic resolution than using shorter sequencing chemistries (i.e., 150bp)4. Using less than 2x300 cycles will result in reads with insufficient overlap and lower quality, thereby reducing usable data. Use >5% PhiX control for low diversity libraries.5,6 References 1. MacConaill, L.E., Burns, R.T., Nag, A. et al. Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing. BMC Genomics 19, 30 (2018). https://doi.org/10.1186/s12864-017-4428-5 2. https://sapac.support.illumina.com/bulletins/2017/03/best-practices-for-manually-normalizing- library-concentrations.html?langsel=/my/ 3. Vargas-Albores F, Ortiz-Suárez LE, Villalpando-Canchola E, Martínez-Porchas M. Size-variable zone in V3 region of 16S rRNA. RNA Biol. 2017;14(11):1514–1521. doi:10.1080/15476286.2017.1317912 4. https://support.illumina.com/content/dam/illumina- marketing/documents/products/other/16s-metagenomics-faq-1270-2014-003.pdf 5. https://support.illumina.com/content/dam/illumina- support/documents/documentation/system_documentation/miseq/miseq-denature-dilute- libraries-guide-15039740-10.pdf 6. https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s- metagenomic-library-prep-guide-15044223-b.pdf
Appendix A: Barcode Sequences for V1-V3 and V4 Set A Position Index (i7) Index2 (i5) A1 ACGATCAG ATATGCGC A2 TCGAGAGT TGGTACAG A3 CTAGCTCA AACCGTTC A4 ATCGTCTC TAACCGGT A5 TCGACAAG GAACATCG A6 CCTTGGAA CCTTGTAG A7 ATCATGCG TCAGGCTT A8 TGTTCCGT GTTCTCGT A9 ATTAGCCG AGAACGAG A10 CGATCGAT TGCTTCCA A11 GATCTTGC CTTCGACT A12 AGGATAGC CACCTGTT B1 GTAGCGTA ATCACACG B2 AGAGTCCA CCGTAAGA B3 GCTACTCT TACGCCTT B4 CTCTGGAT CGACGTTA B5 AGATCGTC ATGCACGA B6 GCTCAGTT CCTGATTG B7 GTCCTAAG GTAGGAGT B8 TATGGCAC ACTAGGAG B9 TCGGATTC CACTAGCT B10 AACAGCGA ACGACTTG B11 CCAACGAA CGTGTGTA B12 CAGTGCTT GTTGACCT C1 GATCAAGG ACTCCATC C2 TCTTCGAC CAATGTGG C3 ATCGTGGT TTGCAGAC C4 CGGTAATC CAGTCCAA C5 AGTTGTGC ACGTTCAG C6 AATGACGC AACGTCTG C7 TACCGGAT TATCGGTC C8 TTGCAACG CGCTCTAT C9 CACTTCAC GATTGCTC C10 TAGCCATG GATGTGTG C11 ACAGGCAT CGCAATCT C12 AGGTGTTG TGGTAGCT D1 CAGTCACA GATAGGCT D2 TCGATGAC AGTGGATC D3 GAAGTGCT TTGGACGT
D4 CTTCCTTC ATGACGTC D5 CGAACAAC GAAGTTGG D6 AACAACCG CATACCAC D7 ACCTCAGT CTGTTGAC D8 CGTCTTCA TGGCATGT D9 TGCGTAAC ATCGCCAT D10 AACACGCT TTGCGAAG D11 ACTCGATC AGTTCGTC D12 TGAGCTGT GAGCAGTA E1 TACTGCTC ACAGCTCA E2 GACGAACT GATCGAGT E3 CTTCGCAA AGCGTGTT E4 ATGGCGAT GTTACGCA E5 ACATGCCA TGAAGACG E6 GTCAACAG ACTGAGGT E7 GTGGTATG CGGTTGTT E8 CCAACTTC GTTGTTCG E9 GACGTCAT GAAGGAAG E10 ACGTCCAA AGCACTTC E11 GATCCACT GTCATCGA E12 AGCCTATC TGTGACTG F1 AGCTACCA CAACACCT F2 AGATTGCG ATGCCTGT F3 CACACATC CATGGCTA F4 GAGCAATC GTGAAGTG F5 ATAGAGCG CGTTGCAA F6 GACCGATA ATCCGGTA F7 CAGACGTT GCGTCATT F8 CTGAACGT GCACAACT F9 TTGGACTG GATTACCG F10 GTCTGCAA ACCACGAT F11 CCACATTG GTCGAAGA F12 GATGGAGT CCTTGATC G1 AGGTCAAC AAGCACTG G2 TACACACG TTCGTTGG G3 CAAGTCGT TCGCTGTT G4 AGCTAGTG GAATCCGA G5 CTCCTAGT GTGCCATA G6 ACTCCTAC CTTAGGAC G7 CAATCAGG AACTGAGC G8 TCGTGCAT GACGATCT G9 TAACGTCG ATCCAGAG G10 AAGGCGTA AGAGTAGC
G11 TCTTACGG TGGACTCT G12 CGTGTGAT TACGCTAC H1 AACAGGTG GCTATCCT H2 AGTCGAAG GCAAGATC H3 TGGAAGCA ATCGATCG H4 CTCGTTCT CGGCTAAT H5 ACGAGAAC ACGGAACA H6 AAGCCTGA CGCATGAT H7 CTACAAGG TTCCAAGG H8 CGATGTTC CTTGTCGA H9 ACCGGTTA GAGACGAT H10 GAACGGTT TGAGCTAG H11 CTGTACCA ACTCTCGA H12 GCGCATAT CTGATCGT
Appendix B: Barcode Sequences for V1-V3 and V4 Set B Position Index (i7) Index2 (i5) A1 TGATAGGC CGACCATT A2 CATCCAAG GATAGCGA A3 GTGAGACT AATGGACG A4 CTGATGAG CGCTAGTA A5 ACGGTACA TCTCTAGG A6 CTCGACTT ACATTGCG A7 ACAACGTG TGAGGTGT A8 TGCTGTGA AATGCCTC A9 CCAAGTAG CTGGAGTA A10 AACTGAGG GTATGCTG A11 AGGTAGGA TGGAGAGT A12 TTCGCCAT CGATAGAG B1 CAGGTAAG CTCATTGC B2 GTATCGAG ACCAGCTT B3 TTCACGGA GAATCGTG B4 GAGCTCTA AGGCTTCT B5 GTCAGTCA CAGTTCTG B6 CACGTCTA TTGGTGAG B7 AATTCCGG CATTCGGT B8 TCTAGGAG TGTGAAGC B9 ATCCGTTG TAAGTGGC B10 GATAGCCA ACGTGATG B11 TATGACCG GTAGAGCA B12 CGATTGGA GTCAGTTG C1 ACAAGCTC ATTCGAGG C2 GAACCTTC GATACTGG C3 AGCGAGAT GCCTTGTT C4 CCGTAACT TTGGTCTC C5 TCAGACAC CCGACTAT C6 CGAAGTCA GTCCTAAG C7 GTGATCCA ACCAATGC C8 ACTGGTGT GATGCACT C9 CTAACCTG GCTGGATT C10 AGCCAACT ATGGTTGC C11 CCAGTTGA CAGAATCG C12 AAGTGCAG GAACGCTT D1 AACCGTGT TCGAACCA D2 CGCGTATT CTATCGCA D3 AGTTCGCA TACGGTTG
D4 TAGTCAGC GAGATGTC D5 AACACCAC CTTACAGC D6 GTAAGCAC AGGAGGAA D7 GTCCTTGA GACGAATG D8 CAGGTTCA GAAGAGGT D9 CCAACACT CGTCAATG D10 GAGAGTAC TACCAGGA D11 AGATACGG CGTACGAA D12 GTTCTTCG GACTTAGG E1 ATTCCGCT AGTGCAGT E2 AAGCTCAC TTGATCCG E3 TGATCACG TGCCATTC E4 CAATGCGA CTTGCTGT E5 ATGCGTCA CCTACTGA E6 TACATCGG CCAAGTTG E7 ACTGCGAA TGATCGGA E8 TCTGTCGT TAGTTGCG E9 CTCAAGCT GTCTGATC E10 AACCACTC CGTTATGC E11 CTTACAGC GCTCTGTA E12 AGTCTTGG TTACCGAG F1 CACGCAAT GCCATAAC F2 AGCTTCAG CTCAGAGT F3 CCTCGTTA CGAGACTA F4 TGAGACGA TGTGCGTT F5 CACAGGAA TTCAGGAG F6 ACTCAACG GACTATGC F7 AAGCGACT AGGTTCGA F8 CCTACCTA AGTCTGTG F9 ATCTCCTG ACCTAAGG F10 TCACGATG TGCAGGTA F11 CCACAACA AAGGACAC F12 AGGTCTGT CAACCTAG G1 AGAAGGAC CTGACACA G2 GCGTATCA ACTCGTTG G3 CAACACAG AGCTCCTA G4 TCCACGTT TACATCGG G5 ATCGCAAC CACAAGTC G6 ACGTCGTT CGGATTGA G7 CGAATACG AGTCGACA G8 TGCTTGCT GTCTCCTT G9 CTCGAACA GAGATACG G10 ACATGGAG ATCGGTGT
G11 ACAAGACG TCTCGCAA G12 CGCCTTAT TCTAACGC H1 AGCAGACA CAATCGAC H2 GTTAAGCG GAGGACTT H3 CATGGATC TGGAGTTG H4 ACAGAGGT CTAGGCAT H5 TAAGTGGC CTCTACTC H6 AGTCAGGT AGAAGCGT H7 GCCTTAAC TCGAAGGT H8 GTTGGCAT GTCGGTAA H9 CAACCTCT ACGATGAC H10 TGGATGGT TCCGTATG H11 CTATCCAC CTAGGTGA H12 GATCTCAG CATTGCCT
Appendix C: Barcode Sequences for V4 Set C Position Index (i7) Index2 (i5) A1 GAACGAAG ACCTTCTC A2 ACCTAGAC TCGTGGAT A3 TACGACGT GTTCATGG A4 TTGAGCTC TAGGATGC A5 AGTACACG CATGGAAC A6 TGTCAGTG GCTTAGCT A7 GACTACGA CTAACTCG A8 TTACGTGC ACCATGTG A9 ACTGCTTG TCAGACGA A10 GCCTATGT TATCAGCG A11 GTACCACA AGCAGATG A12 TAGTGGTG AACGGTCA B1 ATACGCAG CGAACTGT B2 AAGACCGT TCCGAGTT B3 CTCCAATC TTCTCTCG B4 TCTGGACA ATTCTGGC B5 AACACTGG ACTGCTAG B6 TTGGTGCA CATAACGG B7 CCTGTCAA CAGTCTTC B8 CTATGCCT TGCCTCTT B9 TTCGGCTA ACTGTGTC B10 ACCGACAA GTATTGGC B11 CGTAGATG CGATGCTT B12 CTGTATGC AAGGCTGA C1 GTTGCTGT AGTCAGGA C2 AGAACCAG CAGGTATC C3 GATGTCGA TCTCCGAT C4 AGGAGGTT TTCAGCCT C5 AATCGCTG TCTGAGAG C6 AGTGACCT TTAGGTCG C7 CGAATTGC CTCTGGTT C8 CAAGAAGC GCGTTCTA C9 CACCAGTT TCACGTTC C10 GTATTCCG AGGATGGT C11 TTCGAAGC GTGTTCCT C12 AGACCTTG GTAGCATC D1 CCAAGGTT AGGATCTG D2 ACGTATGG GACAAGAG D3 AAGGACCA TTACGGCT
D4 TATGCGGT GCTGTTGT D5 AAGGAAGG AACCGAAG D6 AGCGTGTA TCTGCTCT D7 TCTACGCA CTCAGCTA D8 TGGCTCTT CTTCACCA D9 CCTTCCAT GATCGTAC D10 ATACTGGC CTACAGTG D11 AACCTACG TCGAGTGA D12 CATACTCG CAAGTGCA E1 TGCACTTG CGAGTATG E2 TCACTCGA CGTAGGTT E3 CACTGTAG GCCAGTAT E4 GTACGATC ATGGAAGG E5 TGGTGAAG AAGAGCCA E6 TAGCTGAG TGCGTAGA E7 AGAGCAGA TACACGCT E8 CTTCGGTT CCTTCCTT E9 ACAACAGC ACCGCATA E10 AGCCGTAA TGGTCCTT E11 CTCTTGTC CCATACGT E12 CAGATCCT AACCTTGG F1 GATGCTAC CAAGGTCT F2 AGGAACAC GCTTCGAA F3 ACCATCCT CGGAATAC F4 GAACGTGA AACTGGTG F5 TAGAACGC GCTTCTTG F6 AACCAGAG GCAATTCG F7 CGACCTAA AGGTCACT F8 CTCTCAGA CAGCGATT F9 AGGCTGAA AACCTCCT F10 ATCGGAGA TCGACATC F11 GATACCTG CTGGTTCT F12 TCCTGACT ACAGCAAC G1 TCAGCCTT GCATACAG G2 AAGCATCG CATCTACG G3 GCCAATAC TTGTCGGT G4 GACACAGT TAGCCGAA G5 AAGAGGCA AGGCATAG G6 GAAGACTG TTGACAGG G7 CCGTTATG TGCACCAA G8 CTAGCAGT CCAGTGTT G9 GCCAGAAT TGTCCAGA G10 CGAGAGAA GATTGGAG
G11 AACTCGGA ACGGTCTT G12 ACAGTTCG CTGCGTAT H1 TGACCGTT CACCACTA H2 CATCTGCT TGTGGTAC H3 CGCTGATA ACATAGGC H4 TCGTCTGA CAAGCAGT H5 CACATGGT GCACGTAA H6 CGAGTTAG TCGTAGTC H7 AGCTAAGC CACTGACA H8 GTTCCATG CGTGTACT H9 GCATCCTA GAGCTCAA H10 CCATGAAC ACGTCGTA H11 ATCCACGA GTCTAGGT H12 GAGAAGGT CTTCGTTC
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