Brandon:LabNotes/Project1/2015-8-3
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Update of THS-seq protocol sequences to use Nextera XT V2 sequences
- THS-seq libraries generated with Testing new method of RNA processing, No RNase III, No polyA polymerase (PAP) protocol have not been working on the MiSeq. Cluster density is really low, and when spiked in is 10-20X less then the total number of reads expected to be generated.
- PCR curves go up as expected, gels, gels size selection, RNA quants are all as expected. Tried replacing all reagents for RNA processing. Tried fresh GM12878 samples. Nothing got it to cluster on MiSeq.
- 2015-6-3 THS miseq troubleshooting R1
- 2015-6-10 THS miseq troubleshooting R2
- 2015-6-18 THS miseq troubleshooting R3
- 2015-7-1 THS miseq troubleshooting R4
- 2015-7-14 THS miseq troubleshooting R5
- However, these libraries worked on HiSeq, and 2015-04-01 did not work on miseq
- 2015-3-23 Guanidine HCl after transposition, taq2X and IVT on beads, 100 cell samples
- 2015-4-1 Tn5-059 versus ez-tn5 with THS-seq, using 500 cells and guanidine HCl method
- Called illumina and they said it is either sequencer not being cleaned right, or concentrations are off, or sequences in library generation protocol are not compatible. Considering currently old nextera sequences are being used from 5 years ago, and all current systems illumina sequencing systems do not use them, then maybe that is the problem.
- Thus will redesign THS-seq using Nextera XT V2 sequencing/index/flowcell/amplification primers.
Sequences information, Using T7-nXTv2_i5-1-top and T7-nXTv2_i5-2-top
Sequences: ORIGINAL T7tspn-top2: (69 bp) 5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’ (T7tspn-top2) spcr T7 forward required spcr read sequence spcr ME NEXTERA XT V2 R1 TOP (64 bp) 5'- [CATGG][AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’ (T7-nXTv2_i5-1-top) \---/ \-/\---------------------/ \--------------------------------/ spcr T7 rec. T7 req. forward read seq. \-----------------/ ME NEXTERA XT V2 R1 TOP + EXTRA (69 bp) 5'- [CATGG][AATTAATACGACTCACTATAGGGAGA][CTCCT][TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’ (T7-nXTv2_i5-2-top) \---/ \-/\---------------------/ \---/ \--------------------------------/ spcr T7 rec. T7 req. forward spcr read seq. \-----------------/ ME 5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-T7tspn-bot) T7 description: +1 [CATGG][AAT][TAATACGACTCACTATA|G|GGAGA] [end sequence] rec. T7 req --> [front sequence for optimal binding] After Annealing: 5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’ (T7-nXTv2-1-top) 3'- TCTACACATATTCTCTGTC [Phos]-5' (P-T7tspn-bot) 5'- [phos] CTGTCTCTTATACACATCT -3' 3’- GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’ Fragments created after transposition: 5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNN--------- 5P/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5P ---------NNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’ Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom: 5'- NNN--------- 5P/CTGTCTCTTATACACATCT -3' 5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN -3' 3'- NNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’ 3'- TCTACACATATTCTCTGTC/5P ---------NNN -5' After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also) 5'- CATGG[AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3' 3'- GTACC[TTAATTATGCTGAGTGATATCCCTCT]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNN)NNN -5' 5'- NNN(NNNNNNNNN) CTGTCTCTTATACACATCTGACGCTGCCGACGA[TCTCCCTATAGTGAGTCGTATTAATT]CCATG -3' 3'- NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[AGAGGGATATCACTCAGCATAATTAA]GGTAC -5’ After IVT, All RNA strands will have same 5' end. RNA is also purified after IVT. First G in GGG is the +1 site for the beginning of RNA synthesis for T7. for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator) All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix" RNA 5’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3' After single strand cDNA synthesis to form cDNA/RNA hybrid RNA 5’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5' After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer. Extension with BST or klenow exo- or DNAP1 or Taq2x (been using taq2x) DNA 5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' (Tm=70, anneal@64C) (P-sss_nXTv2_prmr) cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5' Now have double stranded DNA, which should be long. DNA 5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' (Tm=70, anneal@64C) (P-sss_nXTv2_prmr) cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
Custom Nextera i7 transposome i7 adapter 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' (nXTv2_i7_top) 3'- TCTACACATATTCTCTGTC/5Phos/ -5' (P-nXTv2_i7_bot) 5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-nXTv2_i7_bot) 3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nXTv2_i7_top) After custom nextera tagmentation: DNA 5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCT -3' cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' After fill in: DNA 5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' Amplification (to add i5/i7 barcodes): for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator) All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix" 5'- AATGATACGGCGACCACCGA -3' (i5_PCR_ILAdptr, Tm=59C) 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGC -3' (i5XX_T7-1_shrt, comp region Tm=60C, whole=76C) 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTC -3' (i5XX_T7-1_long, comp region Tm=65C, whole=77C) 5’- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' (i5XX_orig, comp region Tm=56C, whole=75C) XXXXXX DNA 5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (i7XX_orig, comp region Tm=57C, whole=73C) 3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' (i7XX_long, comp region Tm=59C, whole=73C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' (i7_PCR_ILAdptr, Tm=58C) 3'- AGCATACGGCAGAAGACGAAC -5' Read Primers: Using i5_shrt or i5_long and i7_long nXT i5 index read -----> nXT read1 -----> nXT i7 index read -----> 5'- AATGATACGGCGACCACCGAGATCTACAC -3' 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' 3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' <----- read2 nXT
primers to order: (T7-nXTv2_i5-1-top) 5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’ (T7-nXTv2_i5-2-top) 5'- CATGGAATTAATACGACTCACTATAGGGAGACTCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’ (P-T7tspn-bot) 5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-sss_nXTv2_prmr) 5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' (nXTv2_i7_top) 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' (P-nXTv2_i7_bot) 5'- /5Phos/CTGTCTCTTATACACATCT -3' (i5_PCR_ILAdptr) 5'- AATGATACGGCGACCACCGA -3' (i7_PCR_ILAdptr) 5'- CAAGCAGAAGACGGCATACGA -3' (i502_T7-1_shrt) 5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGC -3' (i503_T7-1_shrt) 5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGC -3' (i505_T7-1_shrt) 5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGC -3' (i506_T7-1_shrt) 5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGC -3' (i502_T7-1_long) 5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGCGTC -3' (i503_T7-1_long) 5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGCGTC -3' (i505_T7-1_long) 5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGCGTC -3' (i506_T7-1_long) 5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGCGTC -3' (i701_long) 5'- CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG -3' (i702_long) 5'- CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG -3' (i703_long) 5'- CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAG -3' (i704_long) 5'- CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAG -3'
original Nextera XT V2 i5/i7 barcode sequencers and primers num. i5 barcode i5 name i5 smplsheet i5 full sequence 1 CTCTCTAT S502 CTCTCTAT AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC 2 TATCCTCT S503 TATCCTCT AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTC 3 GTAAGGAG S505 GTAAGGAG AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTC 4 ACTGCATA S506 ACTGCATA AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTC 5 AAGGAGTA S507 AAGGAGTA AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTC 6 CTAAGCCT S508 CTAAGCCT AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTC 7 CGTCTAAT S510 CGTCTAAT AATGATACGGCGACCACCGAGATCTACACCGTCTAATTCGTCGGCAGCGTC 8 TCTCTCCG S511 TCTCTCCG AATGATACGGCGACCACCGAGATCTACACTCTCTCCGTCGTCGGCAGCGTC 9 TCGACTAG S513 TCGACTAG AATGATACGGCGACCACCGAGATCTACACTCGACTAGTCGTCGGCAGCGTC 10 TTCTAGCT S515 TTCTAGCT AATGATACGGCGACCACCGAGATCTACACTTCTAGCTTCGTCGGCAGCGTC 11 CCTAGAGT S516 CCTAGAGT AATGATACGGCGACCACCGAGATCTACACCCTAGAGTTCGTCGGCAGCGTC 12 GCGTAAGA S517 GCGTAAGA AATGATACGGCGACCACCGAGATCTACACGCGTAAGATCGTCGGCAGCGTC 13 CTATTAAG S518 CTATTAAG AATGATACGGCGACCACCGAGATCTACACCTATTAAGTCGTCGGCAGCGTC 14 AAGGCTAT S520 AAGGCTAT AATGATACGGCGACCACCGAGATCTACACAAGGCTATTCGTCGGCAGCGTC 15 GAGCCTTA S521 GAGCCTTA AATGATACGGCGACCACCGAGATCTACACGAGCCTTATCGTCGGCAGCGTC 16 TTATGCGA S522 TTATGCGA AATGATACGGCGACCACCGAGATCTACACTTATGCGATCGTCGGCAGCGTC num. i7 barcode i7 name i7 smplsheet i7 full sequence 1 TCGCCTTA N701 TAAGGCGA CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG 2 CTAGTACG N702 CGTACTAG CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG 3 TTCTGCCT N703 AGGCAGAA CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG 4 GCTCAGGA N704 TCCTGAGC CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGG 5 AGGAGTCC N705 GGACTCCT CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGG 6 CATGCCTA N706 TAGGCATG CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGG 7 GTAGAGAG N707 CTCTCTAC CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGG 8 CAGCCTCG N710 CGAGGCTG CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGG 9 TGCCTCTT N711 AAGAGGCA CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGG 10 TCCTCTAC N712 GTAGAGGA CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGG 11 TCATGAGC N714 GCTCATGA CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGG 12 CCTGAGAT N715 ATCTCAGG CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGG 13 TAGCGAGT N716 ACTCGCTA CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGG 14 GTAGCTCC N718 GGAGCTAC CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGG 15 TACTACGC N719 GCGTAGTA CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGG 16 AGGCTCCG N720 CGGAGCCT CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGG 17 GCAGCGTA N721 TACGCTGC CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGG 18 CTGCGCAT N722 ATGCGCAG CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGG 19 GAGCGCTA N723 TAGCGCTC CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGG 20 CGCTCAGT N724 ACTGAGCG CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGG 21 GTCTTAGG N726 CCTAAGAC CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGG 22 ACTGATCG N727 CGATCAGT CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGG 23 TAGCTGCA N728 TGCAGCTA CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGG 24 GACGTCGA N729 TCGACGTC CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGG