|
| Status |
Public on Feb 03, 2019 |
| Title |
Env sCD4 Sorted Library A; Replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
Synthetic coding sequences
|
| Organism |
Human immunodeficiency virus 1 |
| Characteristics |
protein: Env
sort gate: Top 0.5% of population for binding signal (200 nM sCD4(D1-D2))
# collected cells: 259,000
|
| Treatment protocol |
Cells expressing DNA libraries of HIV-1(BaL) Env mutants (cloned into a modified version of pCEP4, Invitrogen, with an upstream chimeric intron) were FACS sorted for binding to different protein/antibody ligands. Three single site-saturation mutagenesis libraries were created, spanning residues 31-265 (Library A; numbering based on the HXB2 reference strain), 266-529 (Library B), and 530-856 (Library C).
|
| Growth protocol |
Expi293F cells cultured in Expi293 Expression Medium (Life Technologies)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was harvested from sorted cells using GeneJet RNA Purification Kit (Thermo Scientific) and cDNA prepared using Accuscript Hi-Fi (Agilent Genomics) primed with a gene-specific primer: BALgp160_libA_RT_rev (CGTTCAGCTGAACAATGATG, Library A), BALgp160_libB_RT_rev (TGTTGGACAATACCAGACAG, Library B), and EBV_reverse (GTGGTTTGTCCAAACTCATC, Library C).
In the first round of PCR (18 cycles), the Env coding sequences were amplified as overlapping fragments of ~800 bp each.
Primer pair (Library A Fragment 1): MiSeq_pCEP4intron_F2 (TCTTTCCCTACACGACGCTCTTCCGATCTACGACTCACTATAGGCTAGC) and MiSeq_BaLgp160_588_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCTGGGTAATCACAGAAGTG)
Primer pair (Library A Fragment 2): MiSeq_BaLgp160_167_F (TCTTTCCCTACACGACGCTCTTCCGATCTACACCGAGGTGCATAATGTG) and MiSeq_BaLgp160_799_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATCACCACCTCTTC)
Primer pair (Library B Fragment 1): MiSeq_BaLgp160_773_F (TCTTTCCCTACACGACGCTCTTCCGATCTGCTGCTGAATGGATCCTTG) and MiSeq_BaLgp160_1368_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTAAACACCTCTGTCTTG)
Primer pair (Library B Fragment 2): MiSeq_BaLgp160_971_F (TCTTTCCCTACACGACGCTCTTCCGATCTTAGACAAGCCCACTGCAAC) and MiSeq_BaLgp160_1579_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCACTGTCAGTGTCATAGC)
Primer pair (Library C Fragment 1): MiSeq_BaLgp160_1492_F (TCTTTCCCTACACGACGCTCTTCCGATCTGTTGTGCAGAGAGAGAAGAG) and MiSeq_BaLgp160_2251_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAACAGGCTTCTCAAGTCCAC)
Primer pair (Library C Fragment 2): MiSeq_BaLgp160_1699_F (TCTTTCCCTACACGACGCTCTTCCGATCTAAACAATTGCAGGCTCGCGT) and MiSeq_BaLgp160_2422_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACAGCTGTGGCATTAAGGAG)
Primer pair (Library C Fragment 3): MiSeq_BaLgp160_1855_F (TCTTTCCCTACACGACGCTCTTCCGATCTATGACGTGGATTGAGTGGGA) and MiSeq_pCEP4_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTATCATGTCTGGATCCGG)
In a second round of PCR (14 cycles), Illumina adaptors and experiment-specific barcodes were added.
Primer pair for adding I5 and I7 Illumina adaptors: MiSeq_Start_Adaptamer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and MiSeq_Index_Adaptamer (CAAGCAGAAGACGGCATACGAGAT-6nt-barcode-GTGACTGGAGTTCAGACGTGTGCTCTTC)
Amplicons were deep sequenced with MiSeq v3 and HiSeq 2500 instruments using a 2x250 nt paired end protocol, though data is analyzed as single reads.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Deep mutational scan of Env for binding to soluble CD4
enrichment_ratios_BaL.xlsx
|
| Data processing |
Data was analyzed using Enrich: http://depts.washington.edu/sfields/software/enrich/
Fuser script: FragmentA1 read 1: python Paired_read_fuser.py --path CD4_fragA1_R1/ --read1 BaLgp160_CD4sort1_A_R1.fastq --read2 BaLgp160_CD4sort1_A_R1.fastq --wtseq GAGGAAAAGCTCTGGGTGACTGTGTATTACGGGGTCCCAGTTTGGAAGGAGGCTACTACCACGCTCTTCTGCGCCTCCGACCGGAAGGCATACGACACCGAGGTGCATAATGTGTGGGCCACACACGCCTGTGTGCCTACC --read1_overlap_start 98 --read1_overlap_end 239 --mode R1
Fuser script: Fragment A1 read 2: python Paired_read_fuser.py --path CD4_fragA1_R2/ --read1 BaLgp160_CD4sort1_A_R2.fastq --read2 BaLgp160_CD4sort1_A_R2.fastq --wtseq TGTACTGACCTCCGGAATGCCACGAACGGAAACGACACAAATACCACGTCAAGCTCCCGAGGGATGGTGGGAGGGGGAGAAATGAAGAACTGCTCATTCAATATAACGACGAATATCCGAGGGAAGGTTCAGAAAGAGTATGCCCTGTTTTACAAACTGGACATCGCTCCTATCGACAACAACAGCAATAACAGGTACCGCCTGATTAGCTGC --read2_overlap_start 15 --read2_overlap_end 228 --mode R2
Fuser script: Fragment A2 read 1: python Paired_read_fuser.py --path CD4_fragA2_R1/ --read1 BaLgp160_CD4sort1_A_R1.fastq --read2 BaLgp160_CD4sort1_A_R1.fastq --wtseq TGGGCCACACACGCCTGTGTGCCTACCGACCCCAACCCACAGGAAGTGGAACTGAAGAACGTGACTGAGAATTTCAATATGTGGAAAAATAATATGGTGGAGCAGATGCACGAGGACATTATTAGCCTCTGGGATCAGTCCCTTAAACCCTGCGTGAAACTGACACCGCTGTGCGTAACGCTGAATTGTACTGACCTCCGGAATGCCACGAACGGAAAC --read1_overlap_start 20 --read1_overlap_end 239 --mode R1
Fuser script: Fragment A2 read 2: python Paired_read_fuser.py --path CD4_fragA2_R2/ --read1 BaLgp160_CD4sort1_A_R2.fastq --read2 BaLgp160_CD4sort1_A_R2.fastq --wtseq TGCAACACTTCTGTGATTACCCAGGCCTGCCCCAAGGTGAGCTTCGAGCCTATCCCAATCCACTACTGCGCCCCAGCAGGCTTTGCCATCCTGAAATGCAAGGACAAGAAGTTCAACGGCAAAGGCCCATGTACAAATGTCAGTACAGTACAGTGCACACACGGTATCCGCCCAGTTGTCTCTACACAACTGCTGCTGAATGGATCCTTG --read2_overlap_start 19 --read2_overlap_end 229 --mode R2
Fuser script: Fragment B1 read 1: python Paired_read_fuser.py --path CD4_fragB1_R1/ --read1 BaLgp160_CD4sort1_B_R1.fastq --read2 BaLgp160_CD4sort1_B_R1.fastq --wtseq GCAGAGGAAGAGGTGGTGATCAGATCTGCTAACTTCGCCGATAATGCTAAAGTCATCATTGTTCAGCTGAACGAGTCTGTCGAAATCAATTGTACTCGCCCAAACAACAACACGCGCAAGAGCATTCATATCGGACCGGGCAGGGCCTTTTACACAACAGGGGAGATTATTGGTGATATTAGACAAGCCCACTGCAACCTCAGTCGAGCAAAGTGGAAT --read1_overlap_start 19 --read1_overlap_end 238 --mode R1
Fuser script: Fragment B1 read 2: python Paired_read_fuser.py --path CD4_fragB1_R2/ --read1 BaLgp160_CD4sort1_B_R2.fastq --read2 BaLgp160_CD4sort1_B_R2.fastq --wtseq ACCCAGCTGTTCAACAGTACGTGGAACGTGACAGAGGAGTCTAACAACACCGTGGAGAATAATACGATCACCCTTCCATGTCGGATTAAGCAGATCATCAATATGTGGCAGGAGGTGGGAAGAGCAATGTACGCGCCGCCCATTAGGGGCCAGATCAGATGCAGTTCAAATATCACAGGCCTGCTCCTGACACGCGACGGAGGCCCAGAGGAC --read2_overlap_start 15 --read2_overlap_end 228 --mode R2
Fuser script: Fragment B2 read 1: python Paired_read_fuser.py --path CD4_fragB2_R1/ --read1 BaLgp160_CD4sort1_B_R1.fastq --read2 BaLgp160_CD4sort1_B_R1.fastq --wtseq CTCAGTCGAGCAAAGTGGAATGATACTCTCAACAAAATTGTTATCAAATTGCGGGAACAGTTTGGGAACAAGACAATCGTATTTAAGCACTCATCCGGAGGCGATCCAGAAATCGTGACACACTCTTTTAACTGCGGTGGAGAATTTTTCTACTGCAACTCCACCCAGCTGTTCAACAGTACGTGGAACGTGACAGAGGAGTCTAACAACACCGTGGAG --read1_overlap_start 19 --read1_overlap_end 238 --mode R1
Fuser script: Fragment B2 read 2: python Paired_read_fuser.py --path CD4_fragB2_R2/ --read1 BaLgp160_CD4sort1_B_R2.fastq --read2 BaLgp160_CD4sort1_B_R2.fastq --wtseq GACAACAAGACAGAGGTGTTTAGGCCCGGCGGCGGCGACATGCGAGATAATTGGAGGTCTGAATTGTACAAGTACAAGGTGGTGAAAATCGAACCCCTGGGAGTGGCGCCTACTAAGGCAAAGCGCCGCGTTGTGCAGAGAGAGAAGAGGGCAGTGGGAATTGGGGCGGTTTTTCTTGGGTTCCTGGGGGCTGCAGGTAGCACC --read2_overlap_start 15 --read2_overlap_end 219 --mode R2
Fuser script: Fragment C1 read 1: python Paired_read_fuser.py --path CD4_fragC1_R1/ --read1 BaLgp160_CD4sort1_C_R1.fastq --read2 BaLgp160_CD4sort1_C_R1.fastq --wtseq ATGGGAGCGGCCGCTATGACACTGACAGTGCAAGCCAGACTCCTGCTGTCTGGTATTGTCCAACAGCAAAACAATTTGCTCCGCGCAATAGAAGCCCAGCAGCACCTGCTCCAACTGACTGTGTGGGGCATTAAACAATTGCAGGCTCGCGTGCTGGCAGTCGAA --read1_overlap_start 75 --read1_overlap_end 240 --mode R1
Fuser script: Fragment C1 read 2: python Paired_read_fuser.py --path CD4_fragC1_R2/ --read1 BaLgp160_CD4sort1_C_R2.fastq --read2 BaLgp160_CD4sort1_C_R2.fastq --wtseq ATAATGATCGTCGGCGGACTCATCGGGCTCCGAATTGTGTTCTCAGTCCTCAGCATTGTGAATAGGGTGCGCCAGGGCTATTCTCCCCTGAGCTTCCAGACCCATCTGCCCTCTAGCCGAGGACCAGATAGGCCTGGTGGGATCGAGGAAGAAGGCGGGGAACGGGACAGGGATAGGAGTGGTCCTCTTGTTAACGGGTTCCTGGCACTTATATGG --read2_overlap_start 14 --read2_overlap_end 230 --mode R2
Fuser script: Fragment C2 read 1: python Paired_read_fuser.py --path CD4_fragC2_R1/ --read1 BaLgp160_CD4sort1_C_R1.fastq --read2 BaLgp160_CD4sort1_C_R1.fastq --wtseq CTGGCAGTCGAACGGTACCTGAGGGATCAACAGCTCCTGGGCATCTGGGGCTGCTCCGGGAAACTCATCTGTACGACAGCGGTCCCTTGGAATGCCAGCTGGTCCAACAAAAGCCTCAACAAGATTTGGGACAATATGACGTGGATTGAGTGGGACAGGGAAATTAACAATTATACCTCAATCATATATTCTCTGATTGAAGAGTCCCAGAATCAG --read1_overlap_start 21 --read1_overlap_end 237 --mode R1
Fuser script: Fragment C2 read 2: python Paired_read_fuser.py --path CD4_fragC2_R2/ --read1 BaLgp160_CD4sort1_C_R2.fastq --read2 BaLgp160_CD4sort1_C_R2.fastq --wtseq GATAGGAGTGGTCCTCTTGTTAACGGGTTCCTGGCACTTATATGGGTGGACTTGAGAAGCCTGTTCCTGTTCTCCTATCATAGGCTCCGGGATCTTCTCCTGATAGTCATGCGGATCGTCGAGCTGTTGGGGCTTGCTGGCGGCTGGGAAGTACTGAAATATTGGTGGAATCTGTTGCAGTATTGGAGCCAGGAACTTAAGAACTCTGCTGTGAGT --read2_overlap_start 14 --read2_overlap_end 230 --mode R2
Fuser script: Fragment C3 read 1: python Paired_read_fuser.py --path CD4_fragC3_R1/ --read1 BaLgp160_CD4sort1_C_R1.fastq --read2 BaLgp160_CD4sort1_C_R1.fastq --wtseq AGGGAAATTAACAATTATACCTCAATCATATATTCTCTGATTGAAGAGTCCCAGAATCAGCAAGAGAAGAATGAGCAGGAATTGTTGGAACTTGATAAATGGGCAAGCCTGTGGAACTGGTTTGACATAACCAAATGGTTGTGGTATATCAAAATCTTCATAATGATCGTCGGCGGACTCATCGGGCTCCGAATTGTGTTCTCAGTCCTCAGCATTGTG --read1_overlap_start 21 --read1_overlap_end 240 --mode R1
Fuser script: Fragment C3 read 2: python Paired_read_fuser.py --path CD4_fragC3_R2/ --read1 BaLgp160_CD4sort1_C_R2.fastq --read2 BaLgp160_CD4sort1_C_R2.fastq --wtseq CTGAAATATTGGTGGAATCTGTTGCAGTATTGGAGCCAGGAACTTAAGAACTCTGCTGTGAGTCTCCTTAATGCCACAGCTGTTGCTGTGGCCGAAGGAACCGATCGAGTTATTGAAGTACTGCAACGAGCAGTTCGCGCTATTCTGCATATCCCACGACGCATTCGGCAGGGGCTGGAGAGAGCTCTCCTG --read2_overlap_start 18 --read2_overlap_end 210 --mode R2
Aligner script: Fragment A1 read 1: python Fused_read_aligner.py --path CD4_fragA1_R1/ --infile BaLgp160_CD4sort1_A_R1.fast_R1_qc1 --referenceDNA GAGGAAAAGCTCTGGGTGACTGTGTATTACGGGGTCCCAGTTTGGAAGGAGGCTACTACCACGCTCTTCTGCGCCTCCGACCGGAAGGCATACGACACCGAGGTGCATAATGTGTGGGCCACACACGCCTGTGTGCCTACC --referenceAA EEKLWVTVYYGVPVWKEATTTLFCASDRKAYDTEVHNVWATHACVPT --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment A1 read 2: python Fused_read_aligner.py --path CD4_fragA1_R2/ --infile BaLgp160_CD4sort1_A_R2.fast_R2_qc1 --referenceDNA TGTACTGACCTCCGGAATGCCACGAACGGAAACGACACAAATACCACGTCAAGCTCCCGAGGGATGGTGGGAGGGGGAGAAATGAAGAACTGCTCATTCAATATAACGACGAATATCCGAGGGAAGGTTCAGAAAGAGTATGCCCTGTTTTACAAACTGGACATCGCTCCTATCGACAACAACAGCAATAACAGGTACCGCCTGATTAGCTGC --referenceAA CTDLRNATNGNDTNTTSSSRGMVGGGEMKNCSFNITTNIRGKVQKEYALFYKLDIAPIDNNSNNRYRLISC --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner script: Fragment A2 read 1: python Fused_read_aligner.py --path CD4_fragA2_R1/ --infile BaLgp160_CD4sort1_A_R1.fast_R1_qc1 --referenceDNA TGGGCCACACACGCCTGTGTGCCTACCGACCCCAACCCACAGGAAGTGGAACTGAAGAACGTGACTGAGAATTTCAATATGTGGAAAAATAATATGGTGGAGCAGATGCACGAGGACATTATTAGCCTCTGGGATCAGTCCCTTAAACCCTGCGTGAAACTGACACCGCTGTGCGTAACGCTGAATTGTACTGACCTCCGGAATGCCACGAACGGAAAC --referenceAA WATHACVPTDPNPQEVELKNVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDLRNATNGN --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment A2 read 2: python Fused_read_aligner.py --path CD4_fragA2_R2/ --infile BaLgp160_CD4sort1_A_R2.fast_R2_qc1 --referenceDNA TGCAACACTTCTGTGATTACCCAGGCCTGCCCCAAGGTGAGCTTCGAGCCTATCCCAATCCACTACTGCGCCCCAGCAGGCTTTGCCATCCTGAAATGCAAGGACAAGAAGTTCAACGGCAAAGGCCCATGTACAAATGTCAGTACAGTACAGTGCACACACGGTATCCGCCCAGTTGTCTCTACACAACTGCTGCTGAATGGATCCTTG --referenceAA CNTSVITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGKGPCTNVSTVQCTHGIRPVVSTQLLLNGSL --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner script: Fragment B1 read 1: python Fused_read_aligner.py --path CD4_fragB1_R1/ --infile BaLgp160_CD4sort1_B_R1.fast_R1_qc1 --referenceDNA GCAGAGGAAGAGGTGGTGATCAGATCTGCTAACTTCGCCGATAATGCTAAAGTCATCATTGTTCAGCTGAACGAGTCTGTCGAAATCAATTGTACTCGCCCAAACAACAACACGCGCAAGAGCATTCATATCGGACCGGGCAGGGCCTTTTACACAACAGGGGAGATTATTGGTGATATTAGACAAGCCCACTGCAACCTCAGTCGAGCAAAGTGGAAT --referenceAA AEEEVVIRSANFADNAKVIIVQLNESVEINCTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHCNLSRAKWN --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment B1 read 2: python Fused_read_aligner.py --path CD4_fragB1_R2/ --infile BaLgp160_CD4sort1_B_R2.fast_R2_qc1 --referenceDNA ACCCAGCTGTTCAACAGTACGTGGAACGTGACAGAGGAGTCTAACAACACCGTGGAGAATAATACGATCACCCTTCCATGTCGGATTAAGCAGATCATCAATATGTGGCAGGAGGTGGGAAGAGCAATGTACGCGCCGCCCATTAGGGGCCAGATCAGATGCAGTTCAAATATCACAGGCCTGCTCCTGACACGCGACGGAGGCCCAGAGGAC --referenceAA TQLFNSTWNVTEESNNTVENNTITLPCRIKQIINMWQEVGRAMYAPPIRGQIRCSSNITGLLLTRDGGPED --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner script: Fragment B2 read 1: python Fused_read_aligner.py --path CD4_fragB2_R1/ --infile BaLgp160_CD4sort1_B_R1.fast_R1_qc1 --referenceDNA CTCAGTCGAGCAAAGTGGAATGATACTCTCAACAAAATTGTTATCAAATTGCGGGAACAGTTTGGGAACAAGACAATCGTATTTAAGCACTCATCCGGAGGCGATCCAGAAATCGTGACACACTCTTTTAACTGCGGTGGAGAATTTTTCTACTGCAACTCCACCCAGCTGTTCAACAGTACGTGGAACGTGACAGAGGAGTCTAACAACACCGTGGAG --referenceAA LSRAKWNDTLNKIVIKLREQFGNKTIVFKHSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWNVTEESNNTVE --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment B2 read 2: python Fused_read_aligner.py --path CD4_fragB2_R2/ --infile BaLgp160_CD4sort1_B_R2.fast_R2_qc1 --referenceDNA GACAACAAGACAGAGGTGTTTAGGCCCGGCGGCGGCGACATGCGAGATAATTGGAGGTCTGAATTGTACAAGTACAAGGTGGTGAAAATCGAACCCCTGGGAGTGGCGCCTACTAAGGCAAAGCGCCGCGTTGTGCAGAGAGAGAAGAGGGCAGTGGGAATTGGGGCGGTTTTTCTTGGGTTCCTGGGGGCTGCAGGTAGCACC --referenceAA DNKTEVFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGAVFLGFLGAAGST --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner script: Fragment C1 read 1: python Fused_read_aligner.py --path CD4_fragC1_R1/ --infile BaLgp160_CD4sort1_C_R1.fast_R1_qc1 --referenceDNA ATGGGAGCGGCCGCTATGACACTGACAGTGCAAGCCAGACTCCTGCTGTCTGGTATTGTCCAACAGCAAAACAATTTGCTCCGCGCAATAGAAGCCCAGCAGCACCTGCTCCAACTGACTGTGTGGGGCATTAAACAATTGCAGGCTCGCGTGCTGGCAGTCGAA --referenceAA MGAAAMTLTVQARLLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVE --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment C1 read 2: python Fused_read_aligner.py --path CD4_fragC1_R2/ --infile BaLgp160_CD4sort1_C_R2.fast_R2_qc1 --referenceDNA ATAATGATCGTCGGCGGACTCATCGGGCTCCGAATTGTGTTCTCAGTCCTCAGCATTGTGAATAGGGTGCGCCAGGGCTATTCTCCCCTGAGCTTCCAGACCCATCTGCCCTCTAGCCGAGGACCAGATAGGCCTGGTGGGATCGAGGAAGAAGGCGGGGAACGGGACAGGGATAGGAGTGGTCCTCTTGTTAACGGGTTCCTGGCACTTATATGG --referenceAA IMIVGGLIGLRIVFSVLSIVNRVRQGYSPLSFQTHLPSSRGPDRPGGIEEEGGERDRDRSGPLVNGFLALIW --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner script: Fragment C2 read 1: python Fused_read_aligner.py --path CD4_fragC2_R1/ --infile BaLgp160_CD4sort1_C_R1.fast_R1_qc1 --referenceDNA CTGGCAGTCGAACGGTACCTGAGGGATCAACAGCTCCTGGGCATCTGGGGCTGCTCCGGGAAACTCATCTGTACGACAGCGGTCCCTTGGAATGCCAGCTGGTCCAACAAAAGCCTCAACAAGATTTGGGACAATATGACGTGGATTGAGTGGGACAGGGAAATTAACAATTATACCTCAATCATATATTCTCTGATTGAAGAGTCCCAGAATCAG --referenceAA LAVERYLRDQQLLGIWGCSGKLICTTAVPWNASWSNKSLNKIWDNMTWIEWDREINNYTSIIYSLIEESQNQ --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment C2 read 2: python Fused_read_aligner.py --path CD4_fragC2_R2/ --infile BaLgp160_CD4sort1_C_R2.fast_R2_qc1 --referenceDNA GATAGGAGTGGTCCTCTTGTTAACGGGTTCCTGGCACTTATATGGGTGGACTTGAGAAGCCTGTTCCTGTTCTCCTATCATAGGCTCCGGGATCTTCTCCTGATAGTCATGCGGATCGTCGAGCTGTTGGGGCTTGCTGGCGGCTGGGAAGTACTGAAATATTGGTGGAATCTGTTGCAGTATTGGAGCCAGGAACTTAAGAACTCTGCTGTGAGT --referenceAA DRSGPLVNGFLALIWVDLRSLFLFSYHRLRDLLLIVMRIVELLGLAGGWEVLKYWWNLLQYWSQELKNSAVS --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner script: Fragment C3 read 1: python Fused_read_aligner.py --path CD4_fragC3_R1/ --infile BaLgp160_CD4sort1_C_R1.fast_R1_qc1 --referenceDNA AGGGAAATTAACAATTATACCTCAATCATATATTCTCTGATTGAAGAGTCCCAGAATCAGCAAGAGAAGAATGAGCAGGAATTGTTGGAACTTGATAAATGGGCAAGCCTGTGGAACTGGTTTGACATAACCAAATGGTTGTGGTATATCAAAATCTTCATAATGATCGTCGGCGGACTCATCGGGCTCCGAATTGTGTTCTCAGTCCTCAGCATTGTG --referenceAA REINNYTSIIYSLIEESQNQQEKNEQELLELDKWASLWNWFDITKWLWYIKIFIMIVGGLIGLRIVFSVLSIV --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment C3 read 2: python Fused_read_aligner.py --path CD4_fragC3_R2/ --infile BaLgp160_CD4sort1_C_R2.fast_R2_qc1 --referenceDNA CTGAAATATTGGTGGAATCTGTTGCAGTATTGGAGCCAGGAACTTAAGAACTCTGCTGTGAGTCTCCTTAATGCCACAGCTGTTGCTGTGGCCGAAGGAACCGATCGAGTTATTGAAGTACTGCAACGAGCAGTTCGCGCTATTCTGCATATCCCACGACGCATTCGGCAGGGGCTGGAGAGAGCTCTCCTG --referenceAA LKYWWNLLQYWSQELKNSAVSLLNATAVAVAEGTDRVIEVLQRAVRAILHIPRRIRQGLERALL --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
MapCounts script: Example: python mapCounts.py --path directory_fragA1_R1/ --infile BaLgp160_libraryA_R1.fast_R1_qc1_PRO_qc2
MapRatios script: Example: python mapRatios.py --path ratios_directory/ --templatepath /deepseq_scripts/r_deepseq_scripts/ --infile2 mapcounts_library --infile1 mapcounts_sorted
MapParts script: Example: python mapParts.py --path ratios_directory/ --infile mapratios_sorted_library --mode mutations:1
Unlink script: Example: python mapUnlink.py --path ratios/ --infile mapratios_sorted_library.m1 --type protein --mode ratios --size ##
In Excel, the log(base2) enrichment ratio of the wildtype sequence was subtracted from the log(2) enrichment ratios for all single mutations.
Data was then assembled from the different fragments/reads to span the full sequence in Excel. Where there was overlap, real enrichment ratios were averaged and converted back to log(base2). Maximum depletion was set to log(base2) enrichment ratio = -4.
Genome_build: Not applicable
Supplementary_files_format_and_content: Excel spreadsheet of log(base2) enrichment ratios for each single amino acid substitution. Also includes the frequency of each mutation in the naïve plasmid library.
|
| |
|
| Submission date |
Aug 04, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Erik Procko |
| E-mail(s) |
procko@illinois.edu
|
| Phone |
217-300-1454
|
| Organization name |
University of Illinois
|
| Department |
Biochemistry
|
| Lab |
RAL 318G
|
| Street address |
601 S Goodwin Ave
|
| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
|
| Platform ID |
GPL23868 |
| Series (1) |
| GSE102276 |
Mutational tolerance of HIV-1 Env in the CD4, VRC01 and PG16 bound states |
|
| Relations |
| BioSample |
SAMN07450872 |
| SRA |
SRX3062762 |
