Data Analysis Guide | Apollo 324TM NGS Library Prep System PrepXTM mRNA and Illumina® Sequencing Workflow & Data Analysis Guide 1 PrepXTM mRNA Strand Specific Library Prep Worklow 3’ SR Adaptor 5’ rAppAG ATC GGA AGA GCA CAC GTC T-NH2 3’ 3’ SR RT Primer 5’ AGA CGT GTG CTC TTC CGA TCT 3’ 5’ SR Adaptor 5’ rGrUrU rCrArG rArGrU rUrCrU rArCrA rGrUrC rCrGrA rCrGrA rUrC 3’ SR Primer 5’ AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC G-s-A 3’ Index Primer 5’ CAA GCA GAA GAC GGC ATA CGA GAT CGT GAT GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC-s-T 3’ ► The ‘r’ denotes RNA base. ► The ‘s’ denotes 3’ phosphothioate modification. ► Red underlined bases are the index. ► Our kit includes the 3’ SR Adaptor, 3’ SR RT Primer, 5’ SR Adaptor, SP Primer, and one Index Primer with the barcode CGT GAT. ► An additional 47 index primers can be downloaded from our website at: http://www.wafergen.com/support/documentation/PrepX_RNASeq_Index_Primers.PDF mRNA 1.1 mRNA fragmentation with RNase III 1.2 3’ Adaptor ligation Fragmented mRNA 3’ SR Adaptor 3’ SR Adaptor ligated mRNA AAGATCGGAAGAGCACACGTCT 3’ 5’ 1.3 3’ SR RT Primer 3’ SR RT Primer hybridization 3’ SR RT Primer hybridized mRNA AAGATCGGAAGAGCACACGTCT 3’ TCTAGCCTTCTCGTGTGCAGA 5’ 5’ 3’ 1.4 5’ SR Adaptor 5’ SR Adaptor ligation 5’ Adaptor ligated mRNA 5’ GUUCAGAGUUCUACAGUCCGACGAUC 3’ AAGATCGGAAGAGCACACGTCT 3’ TCTAGCCTTCTCGTGTGCAGA 5’ 1.5 Reverse transcription cDNA 3’ CAAGTCTCAAGATGTCAGGCTGCTAG T TCTAGCCTTCTCGTGTGCAGA 5’ This document is designed to provide quick reference information only. For technical support, please contact [email protected]. | Page 1 Data Analysis Guide | Apollo 324TM NGS Library Prep System PrepXTM mRNA and Illumina® Sequencing Workflow & Data Analysis Guide 1 PrepXTM mRNA strand-specific library workflow (continued) cDNA T TCTAGCCTTCTCGTGTGCAGA 5’ 3’ CAAGTCTCAAGATGTCAGGCTGCTAG 1.6 SR Primer SR Primer ligation SR Primer ligated cDNA T TCTAGCCTTCTCGTGTGCAGA 5’ 3’ CAAGTCTCAAGATGTCAGGCTGCTAG 3’ 5’ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA 1.7 Extension Extended double stranded cDNA T TCTAGCCTTCTCGTGTGCAGA 5’ AAGATCGGAAGAGCACACGTCT 3’ 3’ TTACTATGCCGCTGGTGGCTCTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAG 5’ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC SR Primer + 1.8 Index Primer Denature then SR- and Index-Primer ligations SR Primer religated cDNA 3’ TTACTATGCCGCTGGTGGCTCTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAG 5’ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA T TCTAGCCTTCTCGTGTGCAGA 5’ 3’ + Index Primer ligated cDNA 5’ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC AAGATCGGAAGAGCACACGTCT 3’ TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGTAGTGCTAG(18 nt)AAC 5’ 3’ 1.9 Final extension Final PrepXTM cDNA products 3’ TTACTATGCCGCTGGTGGCTCTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAG 5’ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC + 5’ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC 3’ TTACTATGCCGCTGGTGGCTCTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAG T TCTAGCCTTCTCGTGTGCAGA 5’ AAGATCGGAAGAGCACACGTCT 3’ AAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATC(18 nt)TTG 3’ T TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGTAGTGCTAG(18 nt)AAC 5’ This document is designed to provide quick reference information only. For technical support, please contact [email protected]. | Page 2 Data Analysis Guide | Apollo 324TM NGS Library Prep System PrepXTM mRNA and Illumina® Sequencing Workflow & Data Analysis Guide 2 Strand Specific cDNA MiSeq® Bridge Amplification and Sequencing Workflows Indexed cDNA 3’ TTACTATGCCGCTGGTGGCTCTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAG T TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGTAGTGCTAG(18 nt)AAC 5’ 2.1 cDNA hybridized to the flow cell P5 5’ AATGATACGGCGACCACCGA 3’ TTACTATGCCGCTGGTGGCTCTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAG 2.2 cDNA hybridization to flow cell 3’ TTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGTAGTGCTAG(18 nt)AAC 5’ Extension on flow cell Extended cDNA on the flow cell AAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATC(18 nt)TTG 3’ TTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGTAGTGCTAG(18 nt)AAC 5’ 5’ AATGATACGGCGACCACCGA GATCTACACGTTCAGAGTTCTACAGTCCGACGATC 3’ TTACTATGCCGCTGGTGGCT CTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAG 2.3 P7 ligation on flow cell P7 ligated cDNA on the flow cell AAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATC(18 nt)TTG 3’ 5’ AATGATACGGCGACCACCGA GATCTACACGTTCAGAGTTCTACAGTCCGACGATC 18 nt ...ATCTCGTATGCCGTCTTCTGCTTG 3’ TAGAGCATACGGCAGAAGACGAAC 5’ P7 2.4 Extension on flow cell Extended cDNA on the flow cell AAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACG ATC(18 nt)TTG 3’ TTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGTAGTGC TAG(18 nt)AAC 5’ 5’ AATGATACGGCGACCACCGA GATCTACACGTTCAGAGTTCTACAGTCCGACGATC 3’ TTACTATGCCGCTGGTGGCT CTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAG Single end read or paired-end Read 1 Small RNA Sequencing Primer CGACAGGTTCAGAGTTCTACAGTCCGACGATC 3’ TTACTATGCCGCTGGTGGCT CTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAG Paired-end Read 2 5’ AATGATACGGCGACCACCGA GATCTACACGTTCAGAGTTCTACAGTCCGACGATC 2.5 R1 read 2.6 R2 read Flow cell polonization, cleavage on P5, then Read 1 Primer and Indexing Primer hybridizations Multiplexing Index Read Sequencing Primer GATCGGAAGAGCACACGTCTGAACTCCAGTCAC Index read TTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGTAGTGC TAG(18 nt)AAC 5’ Flow cell re-polonization, cleavage on P7, then Read 2 Primer hybridization AAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACG ATC(18 nt)TTG 3’ TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG Multiplexing Read 2 Sequencing Primer This document is designed to provide quick reference information only. For technical support, please contact [email protected]. | Page 3 3’ TTACTATGCCGCTGGTGGCTCTAGATGTGCAAGTCTCAAGATGTCAGGCTGCTAG AATGATACGGCGACCACCGA P5 CGACAGGTTCAGAGTTCTACAGTCCGACGATC Small RNA Sequencing Primer 5’ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC P7 TAGAGCATACGGCAGAAGACGAAC TTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGTAGTGCTAGAGCATACGGCAGAAGACGAAC 5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCAC Multiplexing Index Read Sequencing Primer AAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG 3’ Multiplexing Read 2 Sequencing Primer T TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG Trim point for R1 Read cDNA cDNA cDNA cDNA Trim the first T 2 Trim point for R2 Read Summary Data Analysis Guide | Apollo 324TM NGS Library Prep System PrepXTM mRNA and Illumina® Sequencing Workflow & Data Analysis Guide Stand-specific cDNA Illumina® bridge amplification and sequencing workflows (continued) This document is designed to provide quick reference information only. For technical support, please contact [email protected]. | Page 4 Data Analysis Guide | Apollo 324TM NGS Library Prep System PrepXTM mRNA and Illumina® Sequencing Workflow & Data Analysis Guide This document is designed to provide quick reference information only. For technical support, please contact [email protected]. 7400 Paseo Padre Parkway Fremont, CA 94555 U.S.A. [email protected] +1 510.651.4450 www.wafergen.com Enabling clinical sequencing through complete coverage © Copyright 2014 WaferGen Biosystems, Inc. WaferGen Biosystems and its design are registered trademarks of WaferGen Biosystems, Inc. All other trademarks are the property of their respective owners. Our products and services are subject to certain intellectual property and license restrictions for more information please contact us at: 7400 Paseo Padre Parkway, Fremont, CA 94555 or [email protected]. Some oligonucleotide sequences © 2007-2012 Illumina, Inc. All right reserved. | Page 5
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