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Sample GSM3291083

Query DataSets for GSM3291083

Status

Public on Feb 01, 2019

Title

Env PG16 Sorted Library C; Replicate 2

Sample type

SRA

Source name

Synthetic coding sequences

Organism

Human immunodeficiency virus 1

Characteristics

protein: Env (gp140)
sort gate: Top 0.6% of population for binding signal (3 nM PG16)
# collected cells: 61,000

Treatment protocol

Cells expressing DNA libraries of HIV-1(DU422) gp140 mutants (cloned into pCEP4, Invitrogen) were FACS sorted for binding to soluble CD4 (domains D1-D2) or broadly-neutralizing antibody PG16. The extracellular region of Env (gp140) was fused at its C-terminus vi a gly/ser-rich linker and 6H-tag to a canonical transmembrane helix for surface display. Three single site-saturation mutagenesis libraries were created, spanning residues 31-279 (Library A; numbering based on the HXB2 reference strain), 280-577 (Library B), and 578-677 (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, Libraries A, B and C) or SuperScript IV VILO (Invitrogen, Library A) primed with: random hexamers and oligo-dT (Library A, SuperScript), DU422gp160_libA_RT_rev2 (GGCCTAGTGCATTTAATCTC, Library A, Accuscript), DU422gp160_libB_RT_rev2 (GTTGCTGGTCCTTAAGATAC, Library B), and EBV_reverse (GTGGTTTGTCCAAACTCATC, Library C).
In the first round of PCR, the gp140 coding sequences were amplified as overlapping fragments of ~800 bp each.
Primer pair (Library A Fragment 1): HiSeq_DU422gp160_52_F (TCTTTCCCTACACGACGCTCTTCCGATCTTGTAGCGTCAGTGCTTGC) and HiSeq_DU422gp160_624_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACACTTCAGTATGGCGTACC)
Primer pair (Library A Fragment 2): HiSeq_DU422gp160_279_F (TCTTTCCCTACACGACGCTCTTCCGATCTCATGTGGAAGAACGACATGG) and HiSeq_DU422gp160_850_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACGGACTTGTTGAGATGGAC)
Primer pair (Library B Fragment 1): HiSeq_DU422gp160_808_F (TCTTTCCCTACACGACGCTCTTCCGATCTGCGAAGTGAGAACTTGACGA) and HiSeq_DU422gp160_1489_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCCAACCGCTCGCTTTTC)
Primer pair (Library B Fragment 2): HiSeq_DU422gp160_1035_F (TCTTTCCCTACACGACGCTCTTCCGATCTAACTGCGGGAGCATTACAAC) and HiSeq_DU422gp160_1713_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGATACCGCTCAATCGCGA)
Primer pair (Library C): HiSeq_DU422gp160_1671_F (TCTTTCCCTACACGACGCTCTTCCGATCTGGGGCATAAAGCAACTG) and HiSeq_6His-Linker_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTGATGGTGATGATGTCC)
In a second round of PCR, 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 on a HiSeq 2500 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 HiSeq 2500

Description

Deep mutational scan of DU422 gp140 for binding to broadly-neutralizing antibody PG16

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 fragA1_R1/ --read1 DU422gp140_A_R1.fastq.bz2 --read2 DU422gp140_A_R1.fastq.bz2 --wtseq AATCTCGACCTTTGGGTCACCGTATACTACGGAGTCCCCGTGTGGAAAGAGGCTAAGACGACCCTGTTTTGTGCGTCAGACGCGAAAGCGTATGACAAGGAAGTGCATAACGTGTGGGCAACACACGCATGTGTTCCGACGGACCCTAACCCGCAGGAAATCGTCCTTGAAAACGTGACCGAAAACTTCAACATGTGGAAGAACGACATGGTTGACCAA --read1_overlap_start 19 --read1_overlap_end 238 --mode R1
Fuser script: Fragment A1 read 2: python Paired_read_fuser.py --path fragA1_R2/ --read1 DU422gp140_A_R2.fastq.bz2 --read2 DU422gp140_A_R2.fastq.bz2 --wtseq TCCTTCAACACCACGACAGAACTTAGAGATAAAAAGCAAAAAGTCTACGCCCTTTTCTATAAACCGGATGTCGTGCCACTTAACGGTGGTGAGCACAATGAGACAGGTGAGTACATTCTCATCAACTGCAATTCATCTACAATAACGCAAGCATGTCCGAAAGTTTCCTTCGATCCGATCCCTATTCACTACTGCGCTCCTGCA --read2_overlap_start 25 --read2_overlap_end 229 --mode R2
Fuser script: Fragment A2 read 1: python Paired_read_fuser.py --path fragA2_R1/ --read1 DU422gp140_A_R1.fastq.bz2 --read2 DU422gp140_A_R1.fastq.bz2 --wtseq GACCAAATGCACGAGGACATAATCAGTCTTTGGGATCAATCCTTGAAGCCCTGTGTGAAGCTGACGCCTTTGTGCGTAACTTTGAACTGTAAAAACGTAAATATTTCAGCGAATGCTAACGCGACCGCTACACTTAATTCAAGTATGAACGGCGAAATAAAGAATTGCTCCTTCAACACCACGACAGAACTTAGAGATAAAAAGCAA --read1_overlap_start 22 --read1_overlap_end 229 --mode R1
Fuser script: Fragment A2 read 2: python Paired_read_fuser.py --path fragA2_R2/ --read1 DU422gp140_A_R2.fastq.bz2 --read2 DU422gp140_A_R2.fastq.bz2 --wtseq GCAGGGTACGCCATACTGAAGTGTAACAATAAAACGTTTAACGGAACAGGCCCTTGCAATAATGTAAGTACTGTCCAGTGCACGCATGGCATAAAACCCGTCGTGAGCACACAACTTCTTCTTAACGGATCACTCGCCGAGGAAGAGATCATCGTGCGAAGTGAGAACTTGACGAAT --read2_overlap_start 35 --read2_overlap_end 212 --mode R2
Fuser script: Fragment B1 read 1: python Paired_read_fuser.py --path fragB1_R1/ --read1 DU422gp140_B_R1.fastq.bz2 --read2 DU422gp140_B_R1.fastq.bz2 --wtseq AATATAAAGACGATCATAGTCCATCTCAACAAGTCCGTCGAGATTAAATGCACTAGGCCCAACAACAATACGCGGAAATCCGTACGGATAGGTCCTGGACAAACATTCTATGCTACCGGTGAGATTATAGGTGATATACGCGAAGCTCATTGCAATATCAGTAGGGAGACCTGGAATTCCACTTTGATCCAAGTGAAAGAAAAACTGCGGGAGCATTACAAC --read1_overlap_start 22 --read1_overlap_end 244 --mode R1
Fuser script: Fragment B1 read 2: python Paired_read_fuser.py --path fragB1_R2/ --read1 DU422gp140_B_R2.fastq.bz2 --read2 DU422gp140_B_R2.fastq.bz2 --wtseq CCGCCGATTGAAGGGAATATTACTTGCAAGAGCAACATAACTGGTCTTCTTCTCACATGGGATGGTGGAGAGAACAGTACCGAAGGCGTTTTTAGGCCTGGCGGTGGCAATATGAAGGATAACTGGCGCTCAGAACTCTATAAGTACAAAGTCGTCGAAATAAAGCCCTTGGGCGTTGCGCCTACGAAAAGTAAACGAAAAGTAGTAGGGAGG --read2_overlap_start 18 --read2_overlap_end 231 --mode R2
Fuser script: Fragment B2 read 1: python Paired_read_fuser.py --path fragB2_R1/ --read1 DU422gp140_B_R1.fastq.bz2 --read2 DU422gp140_B_R1.fastq.bz2 --wtseq AAAACGATCAAATTTGAACCTTCTTCTGGAGGTGACCTGGAGGTTACGACGCACAGTTTTAACTGTAGGGGAGAATTCTTCTACTGCGATACGACCAAGCTGTTCAACGAGACTAAGCTGTTTAACGAGTCAGAATACGTTGACAATAAGACTATTATCTTGCCCTGCCGGATAAAGCAAATTATCAATATGTGGCAAGAAGTGGGAAGGGCAATGTACGCG --read1_overlap_start 20 --read1_overlap_end 242 --mode R1
Fuser script: Fragment B2 read 2: python Paired_read_fuser.py --path fragB2_R2/ --read1 DU422gp140_B_R2.fastq.bz2 --read2 DU422gp140_B_R2.fastq.bz2 --wtseq GAAAAGCGAGCGGTTGGCTTGGGTGCAGTGCTTCTCGGGTTCCTGGGAGCTGCGGGGTCAACAATGGGAGCTGCTTCCATAACCTTGACTGTGCAGGCAAGGCAGCTGCTGAGTGGGATAGTGCAGCAACAATCCAATCTGCTTAGGGCAATTGAAGCTCAGCAACATCTTTTGCAGCTCACAGTCTGGGGCATAAAGCAACTGCAA --read2_overlap_start 13 --read2_overlap_end 220 --mode R2
Fuser script: Fragment C read 1: python Paired_read_fuser.py --path fragC_R1/ --read1 DU422gp140_C_R1.fastq.bz2 --read2 DU422gp140_C_R1.fastq.bz2 --wtseq ACCAGAGTCCTCGCGATTGAGCGGTATCTTAAGGACCAGCAACTGCTGGGACTCTGGGGGTGTTCCGGGAAGCTTATTTGTGCCACTGCGGTACCATGGAACTCCTCTTGGTCCAACAAGTCTCTGGGCGATATTTGGGACAATATGACGTGGATGCAGTGGGATCGCGAGATATCCAACTACACCAACACGATCTTCCGGCTT --read1_overlap_start 20 --read1_overlap_end 224 --mode R1
Fuser script: Fragment C read 2: python Paired_read_fuser.py --path fragC_R2/ --read1 DU422gp140_C_R2.fastq.bz2 --read2 DU422gp140_C_R2.fastq.bz2 --wtseq CTGGGCGATATTTGGGACAATATGACGTGGATGCAGTGGGATCGCGAGATATCCAACTACACCAACACGATCTTCCGGCTTCTCGAAGACAGTCAGAATCAACAAGAGAAGAATGAGAAGGATCTGTTGGCCCTTGACAGCTGGAAAAATCTTTGGAATTGGTTTGACATTACTAAC --read2_overlap_start 42 --read2_overlap_end 219 --mode R2
Aligner script: Fragment A1 read 1: python Fused_read_aligner.py --path fragA1_R1/ --infile DU422gp140_A_R1.fast_R1_qc1 --referenceDNA AATCTCGACCTTTGGGTCACCGTATACTACGGAGTCCCCGTGTGGAAAGAGGCTAAGACGACCCTGTTTTGTGCGTCAGACGCGAAAGCGTATGACAAGGAAGTGCATAACGTGTGGGCAACACACGCATGTGTTCCGACGGACCCTAACCCGCAGGAAATCGTCCTTGAAAACGTGACCGAAAACTTCAACATGTGGAAGAACGACATGGTTGACCAA --referenceAA NLDLWVTVYYGVPVWKEAKTTLFCASDAKAYDKEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNDMVDQ --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 fragA1_R2/ --infile DU422gp140_A_R2.fast_R2_qc1 --referenceDNA TCCTTCAACACCACGACAGAACTTAGAGATAAAAAGCAAAAAGTCTACGCCCTTTTCTATAAACCGGATGTCGTGCCACTTAACGGTGGTGAGCACAATGAGACAGGTGAGTACATTCTCATCAACTGCAATTCATCTACAATAACGCAAGCATGTCCGAAAGTTTCCTTCGATCCGATCCCTATTCACTACTGCGCTCCTGCA --referenceAA SFNTTTELRDKKQKVYALFYKPDVVPLNGGEHNETGEYILINCNSSTITQACPKVSFDPIPIHYCAPA --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 fragA2_R1/ --infile DU422gp140_A_R1.fast_R1_qc1 --referenceDNA GACCAAATGCACGAGGACATAATCAGTCTTTGGGATCAATCCTTGAAGCCCTGTGTGAAGCTGACGCCTTTGTGCGTAACTTTGAACTGTAAAAACGTAAATATTTCAGCGAATGCTAACGCGACCGCTACACTTAATTCAAGTATGAACGGCGAAATAAAGAATTGCTCCTTCAACACCACGACAGAACTTAGAGATAAAAAGCAA --referenceAA DQMHEDIISLWDQSLKPCVKLTPLCVTLNCKNVNISANANATATLNSSMNGEIKNCSFNTTTELRDKKQ --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 fragA2_R2/ --infile DU422gp140_A_R2.fast_R2_qc1 --referenceDNA GCAGGGTACGCCATACTGAAGTGTAACAATAAAACGTTTAACGGAACAGGCCCTTGCAATAATGTAAGTACTGTCCAGTGCACGCATGGCATAAAACCCGTCGTGAGCACACAACTTCTTCTTAACGGATCACTCGCCGAGGAAGAGATCATCGTGCGAAGTGAGAACTTGACGAAT --referenceAA AGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIVRSENLTN --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 fragB1_R1/ --infile DU422gp140_B_R1.fast_R1_qc1 --referenceDNA AATATAAAGACGATCATAGTCCATCTCAACAAGTCCGTCGAGATTAAATGCACTAGGCCCAACAACAATACGCGGAAATCCGTACGGATAGGTCCTGGACAAACATTCTATGCTACCGGTGAGATTATAGGTGATATACGCGAAGCTCATTGCAATATCAGTAGGGAGACCTGGAATTCCACTTTGATCCAAGTGAAAGAAAAACTGCGGGAGCATTACAAC --referenceAA NIKTIIVHLNKSVEIKCTRPNNNTRKSVRIGPGQTFYATGEIIGDIREAHCNISRETWNSTLIQVKEKLREHYN --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 fragB1_R2/ --infile DU422gp140_B_R2.fast_R2_qc1 --referenceDNA CCGCCGATTGAAGGGAATATTACTTGCAAGAGCAACATAACTGGTCTTCTTCTCACATGGGATGGTGGAGAGAACAGTACCGAAGGCGTTTTTAGGCCTGGCGGTGGCAATATGAAGGATAACTGGCGCTCAGAACTCTATAAGTACAAAGTCGTCGAAATAAAGCCCTTGGGCGTTGCGCCTACGAAAAGTAAACGAAAAGTAGTAGGGAGG --referenceAA PPIEGNITCKSNITGLLLTWDGGENSTEGVFRPGGGNMKDNWRSELYKYKVVEIKPLGVAPTKSKRKVVGR --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 fragB2_R1/ --infile DU422gp140_B_R1.fast_R1_qc1 --referenceDNA AAAACGATCAAATTTGAACCTTCTTCTGGAGGTGACCTGGAGGTTACGACGCACAGTTTTAACTGTAGGGGAGAATTCTTCTACTGCGATACGACCAAGCTGTTCAACGAGACTAAGCTGTTTAACGAGTCAGAATACGTTGACAATAAGACTATTATCTTGCCCTGCCGGATAAAGCAAATTATCAATATGTGGCAAGAAGTGGGAAGGGCAATGTACGCG --referenceAA KTIKFEPSSGGDLEVTTHSFNCRGEFFYCDTTKLFNETKLFNESEYVDNKTIILPCRIKQIINMWQEVGRAMYA --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 fragB2_R2/ --infile DU422gp140_B_R2.fast_R2_qc1 --referenceDNA GAAAAGCGAGCGGTTGGCTTGGGTGCAGTGCTTCTCGGGTTCCTGGGAGCTGCGGGGTCAACAATGGGAGCTGCTTCCATAACCTTGACTGTGCAGGCAAGGCAGCTGCTGAGTGGGATAGTGCAGCAACAATCCAATCTGCTTAGGGCAATTGAAGCTCAGCAACATCTTTTGCAGCTCACAGTCTGGGGCATAAAGCAACTGCAA --referenceAA EKRAVGLGAVLLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQ --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner script: Fragment C read 1: python Fused_read_aligner.py --path fragC_R1/ --infile DU422gp140_C_R1.fast_R1_qc1 --referenceDNA ACCAGAGTCCTCGCGATTGAGCGGTATCTTAAGGACCAGCAACTGCTGGGACTCTGGGGGTGTTCCGGGAAGCTTATTTGTGCCACTGCGGTACCATGGAACTCCTCTTGGTCCAACAAGTCTCTGGGCGATATTTGGGACAATATGACGTGGATGCAGTGGGATCGCGAGATATCCAACTACACCAACACGATCTTCCGGCTT --referenceAA TRVLAIERYLKDQQLLGLWGCSGKLICATAVPWNSSWSNKSLGDIWDNMTWMQWDREISNYTNTIFRL --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment C read 2: python Fused_read_aligner.py --path fragC_R2/ --infile DU422gp140_C_R2.fast_R2_qc1 --referenceDNA CTGGGCGATATTTGGGACAATATGACGTGGATGCAGTGGGATCGCGAGATATCCAACTACACCAACACGATCTTCCGGCTTCTCGAAGACAGTCAGAATCAACAAGAGAAGAATGAGAAGGATCTGTTGGCCCTTGACAGCTGGAAAAATCTTTGGAATTGGTTTGACATTACTAAC --referenceAA LGDIWDNMTWMQWDREISNYTNTIFRLLEDSQNQQEKNEKDLLALDSWKNLWNWFDITN --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 DU422gp140_A_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 and various controls.

Submission date

Jul 18, 2018

Last update date

Feb 01, 2019

Contact name

Erik Procko

E-mail(s)

procko@illinois.edu

Phone

217-300-1454

Organization name

University of Illinois