Defining kir and hla class I genotypes at Highest Resolution via High-Throughput Sequencing
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with no history of chronic disease, were studied. These sam- ples were selected at random from a larger dataset (n ¼ 500) developed as controls for genome-wide association studies (GWASs) of multiple sclerosis (MIM: 126200). 46 These sam- ples were used because their high-resolution HLA-A, HLA-B, and HLA-C genotypes had been determined by Sanger sequencing. 46 To validate the PING pipeline, we used existing sequence- read data from an additional two sources, described below. To extract KIR-specific sequences from these datasets, we used SAMtools 0.1.18. 35 to identify read pairs that mapped within the KIR region (hg19 coordinates: 19:55,228,188–55,383,188) KIR
copy number and
allele genotype
KFFallele Select probes Generate hit pattern Decode pattern SOS Sort reads per KIR Make sam/bam Final align - vcf New SNPs New combinations (manual check) KFFgc
Gene-specific probes Per-sample probe hits +/- genotype MIRAgc
Align vs. reference Count reads per gene KIR gene +/- Copy number KIR Reads
PING_gc PING_allele Filter Call alleles KIR alleles Whole-Genome Sequencing Target Enrichment + NGS All sequence reads Unmapped sequence reads Mapped KIR sequence reads KIR + Copy number A B Call alleles Data To
Validate ValidaƟon Individuals N (used) Genotype Method
1. IHWG cell lines 97 Enrichment/NGS KIR +/- PCR
KIR copy number Real Ɵme PCR KIR alleles Pyrosequencing KIR novel alleles Cloning/Sanger sequencing HLA alleles PCR-SSOP, and IHWG database 2. West African Trios 90 Enrichment/NGS KIR alleles Pyrosequencing KIR copy number SegregaƟon HLA alleles PCR-SSOP
3. Europeans 188 Enrichment/NGS HLA alleles PCR-SSOP, and Sanger sequencing 4a. KhoeSan 15 (Exome) KIR alleles Pyrosequencing 4b. 1000 Genomes 2,112
(Exome) KIR novel alleles Cloning/Sanger sequencing Figure
1. Pipeline
for Analyzing Sequence Data from Highly Polymorphic and Structurally Divergent KIR Haplotypes (A) The PING (Pushing Immunogenetics to the Next Generation) pipeline has two broad arms and two modules. The first module (PING_gc) determines KIR gene copy numbers, and the second module (PING_allele) determines their
alleles. Within each module are two arms. The first arm (KFFgc and KFFallele) is an analysis in- dependent of any alignment or assembly and uses virtual probes to mine the raw data. The second arm (MIRAgc and SOS) performs filtering and alignment of reads to reference sequences. Thus, copy-num- ber and allele genotypes are each derived by two independent methods. The tech- niques are described fully in the Material
and Methods . (B) Data generated by the
method described herein (1–3) and those obtained from other sources (4). The table shows the number of individuals or cell lines used for validation, the genotyping results to be validated, and the independent labo- ratory method used for this purpose. or an unallocated chromosome 19 region (GenBank: GL000209.1) corresponding to an alternative KIR haplotype. 4. 15 KhoeSan individuals. 47 These indi- viduals had also been genotyped for KIR genes via standard lower- throughput methods
of pyrose-
quencing and Sanger sequencing. 16 5. The 1000 Genomes Project data. 48 All 2,532 of the whole-exome- sequenced individuals described in the May 2013 release were tar- geted. 48
tity of sequence reads for the anal- ysis, we excluded samples if fewer than 25 reads mapped to exon 3 of KIR3DL2 or KIR3DL1/2v (see Ap- pendix A
). 420 of the 1000 Genomes samples were excluded on this basis, and the remaining 2,112 were geno- typed. When previously uncharac- terized KIR alleles were identified in the 1000 Genomes dataset, genomic DNA from the source samples was pur- chased from the Coriell Biorepository for confirmation of their sequence by standard molecular methods. Laboratory Methods Design of Capture Oligonucleotide Probes To account for variation in the gene content of the KIR region, we targeted a panel of independently generated reference KIR haplo- types 6,14
that together represent all of the 13 recognized KIR genes. First, we designed probes against the two complete KIR haplotypes (GenBank: FP089703 and FP089704) that were generated from the PGF cell line, which was the source of the human reference The American Journal of Human Genetics 99, 375–391, August 4, 2016 377
sequence for the KIR region. 6 We used end-to-end tiling with strand swapping to design non-overlapping 80-mer probes to match these reference sequences. We then designed a similar set of probes by using a further 27 complete KIR haplotype se- quences
6,14 and all KIR sequences included in the January 2013 release of the IPD-KIR database. 15 In this second stage, probes that differed by more than three nucleotides from the correspond- ing segment of the initial reference haplotypes were selected for use. We did not mask any repetitive elements in the target haplotypes. The KIR genomic region targeted by the probes is equivalent to that covered by chr19: 55,228,188–55,383,188 (UCSC Genome Browser hg19) and an unmapped chromosome 19 region (GenBank: GL000209.1), which are the two KIR haplo- types present in the hg19 reference genome. In a similar manner to the KIR probes, we designed probes against the alleles of the classic HLA class I genes present in the PGF cell line, which was also the source of the human reference sequence for the HLA re- gion.
1,14 These probes were supplemented with probes designed against the 6,795 HLA class I sequences reported in the January 2013 release of the IPD-HLA database. 15 A total of 10,456 capture probes were used. Preparation of Biotinylated Capture Probes The set of capture oligonucleotides, each one comprising a unique sequence flanked by the common sequences 5 0 -GGTGATTGCG TATCT-3 0 (PTL3) and 5 0 -CATGTCGTGGGAATT-3 0 (PTR3), was syn- thesized by CustomArray. This set of oligonucleotides was pooled and amplified in a single PCR using primers with sequences corre- sponding to PTL3 and PTR3. The PCR comprised 1 3 Titanium Taq buffer (Clontech), 1 mM each of biotin-PTL3 and -PTR3 primers (Integrated DNA Technologies), 0.2 mM dNTPs with 12.5% dUTP (Roche), 1 mL (1 unit) Uracil-DNA Glycosylase (UDG; New England Biolabs), 1 M betaine, 3 mL (15 units) AmpliTherm Polymerase (Epicenter), 0.2 ng of the pool of capture oligonucleotides, and H 2 O added to create a final volume of 100 mL. PCR cycling condi- tions were as follows: 37
C for 10 min, 95 C for 3 min, 95
C for
30 s, 55
C for 30 s, 72 C for 30 s ( 328), 72
C for 10 min, and a hold at 10
C. The biotinylated PCR product (100 mL aliquot) was then bound to streptavidin-coated magnetic beads (Illumina) that had been pre-washed with 100 mL 63 hybridization buffer (HB: 1 M NaCl, 0.5 M phosphate buffer, and 0.05% Tween-20) and suspended in 100 mL 123 HB. The incubation was carried out for 30 min at room temperature in HB and with agitation. The beads, now coated with biotinylated oligonucleotides, were then washed: once with 100 mL 63 HB, twice with 100 mL 0.23 HB, once with 100 mL 0.1 nM NaOH and 100 mL 10 mM EDTA, and lastly, once with 100 mL 0.23 HB. Biotinylated oligonucleotides were eluted from the beads with 0.1 mM EDTA and then concentrated via speed vacuum to a final concentration of 2.5 nM for each capture probe. Library Preparation, Enrichment, and Sequencing The protocol was based on the TruSeq Nano method for library preparation (Illumina). The DNA samples we used are described below. For each sample, 300 ng genomic DNA (as determined by Qubit instrument, Thermo Fisher Sceintific) was sheared into 800 bp fragments with a Covaris S220 instrument (Covaris). The library preparation was then performed according to the manufac- turer’s instructions, whereby 96 unique ‘‘dual index’’ combina- tions were used individually to label the library obtained from each DNA sample, and the following modifications: (1) for clean- ing and size selecting the samples after end repair, 70.2 mL sample purification beads plus 89.8 mL H 2
mL sample, and (2) in the final PCR, the 72
C extension time was changed from 30 to 90 s to account for the 800 bp fragment length. Enrichment of HLA and KIR sequences was performed according to a modified version of the Nextera Rapid Capture Exome enrich- ment protocol (Illumina), a solution-based target-capture assay. The libraries of genomic DNA, indexed uniquely for each sample as described above, were pooled prior to their hybridization with the capture probes. Thus, each hybridization mix (100 mL) con-
tained 96 uniquely indexed sequence libraries (62.5 ng for each li- brary and 6,000 ng in total), 50 pM of each biotinylated capture probe, and HB (CT3 and all subsequent buffers from Illumina). The hybridization mix was incubated at 95
C for 10 min, gradu- ally cooled by 2
C/min to 58 C, and then maintained at 58
C
tained targeted KIR and HLA sequences became specifically hy- bridized to biotinylated capture probes. In the next reaction, 100 mL of streptavidin-coated magnetic beads were used to separate the specific hybridized genomic DNA away from the non-specific un-hybridized genomic DNA. The biotin present in hybrid DNA molecules was bound to strep- tavidin on the beads, leaving the non-specific DNA in solution. The DNA preparation enriched with the targeted KIR and HLA genes was then eluted from the beads. Binding of the hybridiza- tion product to the beads was achieved by 30 min incubation with agitation at 1,000 rpm on a plate shaker at room temperature. To clean the product, we removed the streptavidin beads from solution by using a magnetic separator, mixed them with 200 mL Enrichment Wash Solution (Illumina), and incubated them at 50
C for 30 min. This wash step was repeated. To elute the enriched DNA from the beads, we added 23 mL of elution mix (made from 1.5 mL 2M NaOH plus 28 mL Elution Buffer 1, Illu- mina), incubated for 5 min at room temperature and neutralized with 4
mL Elute Target Buffer 2 (Illumina). The eluted material was then subjected to a second round of enrichment from the hybridization step onward. After the gradual cooling step, the hy- bridization mix was maintained at 58
C for 14–18 hr. An aliquot of 10 mL of the enriched DNA preparation was subject to PCR amplification in a 50 mL reaction mix containing 5 mL of a PCR primer cocktail, 15 mL of resuspension buffer, and 20 mL of Nextera Enrichment Amplification Mix. PCR cycling was per- formed as follows: 98
C for 30 s; 17 cycles of 98 C for 10 s, 60
C
C for 30 s; and a final elongation step at 72
C for
5 min. Amplified material was purified with 40 mL of sample purifi- cation beads and eluted in 30 mL resuspension buffer (Illumina). NGS Strategies Set 1: IHWG Cell Lines. The enriched libraries were sequenced with a HiSeq 2000 instrument and sequencing chemistry (Illumina). Samples were clustered and paired-end sequencing was performed with the TruSeq SBSv3-HS Kit (Illumina). The sequencing read length was 2 3 101 bp. Set 2: Trios and Chimpanzee. The enriched libraries obtained from these samples were sequenced with a HiSeq 2500 instrument and sequencing chemistry (Illumina). The sequencing read length was 2 3 250 bp. These samples were also genotyped for HLA-A, HLA-B, and HLA-C with SSOPs and for KIR3DL1 and KIR3DL2 by pyrose- quencing
23 (see
Appendix A ). Set 3: European Control Samples. These samples were analyzed with a MiSeq instrument (Illumina) with V3 chemistry, and the sequencing read length was 2 3 300 bp. Enrichment Efficiency We estimated enrichment efficiency by mapping unprocessed sequence reads to the human reference sequence (hg19) with 378 The American Journal of Human Genetics 99, 375–391, August 4, 2016 Yüklə 448,46 Kb. Dostları ilə paylaş:
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