sequencing library  (Macrogen)

Journal: NAR Genomics and Bioinformatics

Article Title: Whole-genome sequencing with AVITI and NovaSeq X Plus reveals comparable performance with contextual biases

doi: 10.1093/nargab/lqag053

Figure Lengend Snippet: Comparisons of genome coverage and variant calling. ( a ) Differential coverage across the top 15 most variable genome stratifications. Difference measure relative to mean autosomal coverage for the dataset. ( b ) Candidate small variant calls filtered out by DeepVariant across coverage from 10× to 50×. ( c ) F1-score for variant calling for SNPs and INDELs at different levels of genome coverage, as reported by hap.py. PacBio HiFi DeepVariant calls were used as the truth set. Cell line REH was omitted as HiFi coverage was insufficient. ( d ) Relative F1-score for SNPs and INDELs combined comparing AVITI to NovaSeq X Plus across GIAB stratifications (v3.5). The plot shows the top 15 most variable genome stratifications, split by mean overall coverage range. Bars show the mean difference across the subsampled datasets. TR = tandem repeat, HP = homopolymer, Imp. HP = imperfect HP, N-mer = TR composed of N bp repeated elements.

Article Snippet: PacBio HiFi sequencing libraries for MM1S, OPM2, and KMS12BM were prepared using the same high-molecular-weight gDNA source as the short read libraries and the SMRTbell prep kit 3.0 (Pacific Biosciences, #102-141-700).

Techniques: Variant Assay

Journal: bioRxiv

Article Title: Binding dynamics shape germinal center broadly neutralizing responses to HIV priming

doi: 10.64898/2026.05.12.724749

Figure Lengend Snippet: (A) CH31 precursor adoptive transfer and priming schema. B6.SJL CD45.1+ mice were reconstituted with graded numbers of CD45.2+ CH31 UCA hom/hom dKI B cells ( ; Methods) and primed for 8d with eODGT7 or eODGT8 np’s. (B-D) Flow cytometric analysis of GC recruitment in primed CH31 UCA hom/hom dKI →WT chimeras. (B) GC occupancy, defined as the percentage of GC B cells (B220+CD19+CD38–GL7+) that are donor-derived (CD45.2+) and ‘on-target’ (eODGT bait+, KI HC+LC). (C) Representative gating strategy for calculation of the GC ‘specificity index’ ie, on-target donor GC B cells divided by ‘off-target’ (CD45.1+eODGT-) recipient GC B cells. (D) GC recruitment specificity index (on/off-target ratio) following eODGT7 or eODGT8 np priming. Each point represents one primed chimera. (E–I) 10x paired HC/LC sequencing and SHM analysis of individual GC B cells from primed chimeras reconstituted at low (∼1:10 5 ) or ultra-low (∼1:10 6 ) precursor frequencies, corresponding to physiological human estimates and the lower limit of reproducible reconstitution, respectively , . (E) HC/LC pairing status of single-sorted donor GC B cells, with bona fide (V(D)J sequence verified) on-target clones indicated. Pies show HC/LC pairing composition of donor GC B cells; black slices mark on-target CH31 UCA pairs and center values denote unique cell counts. (F) Total SHM distributions among on-target CH31 clones, with bar graphs showing the fraction of on-target CH31 clones that remain germline versus those acquiring ≥1 aa substitution, and accompanying pie charts stratifying mutated clones by aa substitution number. (G) Frequency of VRC01/CH31-class key V H contact residue substitutions among mutated on-target clones . (H) Frequency and distribution of pre-indel events in CH31 HC rearrangements, stratified by HC region and indel length , . (I) Positional distribution of CH31 HC aa mutations across on-target pairs, with AID hotspot motifs (WRC/GYW and WGCW; W =A/T, R=A/G, Y=C/T) indicated by blocks and previously reported insertion sites denoted by arrows , , . Data in panels (F) and (H) represent pooled 10x Ig-seq single cells from chimeras over two independent experiments (five pools; 18 mice per 1/10 5 -reconstituted group; 6 mice per 1/10 6 -reconstituted group, n =48 total). Data in panels (G) to (I) comprise chimeras reconstituted at physiological (1/105) precursor frequencies. Statistical comparisons used Mann–Whitney U tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant.

Article Snippet: FASTQ files were uploaded from 10x Genomics library sequencing and data was analyzed using the Cell Ranger pipeline ( https://www.10xgenomics.com/support/software/loupe-browser/latest ).

Techniques: Adoptive Transfer Assay, Derivative Assay, Sequencing, Clone Assay, Residue, MANN-WHITNEY

Journal: bioRxiv

Article Title: Binding dynamics shape germinal center broadly neutralizing responses to HIV priming

doi: 10.64898/2026.05.12.724749

Figure Lengend Snippet: (A) Schematic of the CH31 precursor transfer and priming strategy. B6.SJL CD45.1 recipients received purified CD45.2⁺ CH31 UCA hom/hom dKI B cells and, after 24h, resulting CH31 UCA hom/hom dKI →WT chimeras (reconstituted at 1:10 5 precursor frequency) were primed for 8 or 16 days with eOD monomers, low-valency tetramers, or high-valency np’s (poly I:C–formulated), or poly I:C alone, prior to GC B cell recovery for flow cytometric phenotyping and 10x paired Ig seq ( n per group indicated). (B) Flow cytometric quantification of donor-derived, on-target GC B cells (live singlet B220⁺CD19⁺CD38⁻GL7⁺CD45.2⁺eODGT bait⁺) shown as the fraction of total GC B cells at day 8 ( top ) or day 16 ( bottom ). Each dot represents one primed CH31 UCA hom/hom dKI chimera. Significance relative to eODGT7 np was determined by Mann–Whitney U tests. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (C–D) Immunofluorescence validation of enhanced CH31 precursor recruitment and GC persistence following eODGT7 np priming. (C) Representative spleen cryosection images from chimeras primed for 16 days with eODGT7 or eODGT8 np’s; boxed regions indicate GC closeups. Note the persistently high fraction of donor CH31 UCA hom/hom dKI (CD45.2⁺) B cells in individual GCs across the spleen (top) of an eODGT7 np–immunized animal at this later time point. (D) Quantification of donor CH31 GC occupancy (CD45.2⁺ area within GL7⁺ GCs) at days 8 and 16, shown as violin plots with medians (blue bars). Each dot represents one GC. Data are pooled from three mice per group. Total GCs analyzed were 698/465 (eODGT7, d8/d16) and 505/325 (eODGT8, d8/d16). Mann–Whitney test; ****p<0.0001. (E-F) SHM magnitude, positional distribution in, and kinetics of, CH31-derived V(D)J rearrangements following eODGT7 np priming, compared with eODGT8 np or eODGT8 tetramer priming. GC B cells were isolated by flow sorting and analyzed by paired 10x Ig-seq. (E) Pie charts show SHM distributions among bona fide on-target CH31 UCA HC/LC pairs, with slices indicating total aa substitutions. ≥1000 unique, non-oligoclonal sequences from ≥5 pooled mice per group were analyzed. Data from eODGT8 np–primed mice are omitted due to insufficient recovered precursors. (F) Frequency of CH31 UCA HC aa mutations by residue position among all on-target pairs. AID hotspots (WRC/GYW and WGCW) are indicated by pink blocks. Previously reported insertion sites from bnAb lineage retracement , or vaccine-induced maturation are marked by arrows. (G) T follicular helper (T fh ) cell responses in chimeras primed for 8 or 16 days with eODGT7 or eODGT8 np’s, or eODGT8 tetramers. Shown is the percentage of live, singlet splenocytes that were T fh (CD4⁺CD44⁺CD62L⁻PD1⁺CXCR5⁺CD25⁻CD127⁺) at peak GC occupancy (d8) or peak SHM (d16). Gating strategy is shown in Fig S16.

Article Snippet: FASTQ files were uploaded from 10x Genomics library sequencing and data was analyzed using the Cell Ranger pipeline ( https://www.10xgenomics.com/support/software/loupe-browser/latest ).

Techniques: Purification, Cell Recovery, Derivative Assay, MANN-WHITNEY, Immunofluorescence, Biomarker Discovery, Isolation, Residue