Quantifying Hematopoietic Stem Cell Clonal Diversity by Selecting Informative Amplicon Barcodes

Abstract

Hematopoietic stem cells (HSCs) are functionally and genetically diverse and this diversity decreases with age and disease. Numerous systems have been developed to quantify HSC diversity by genetic barcoding, but no framework has been established to empirically validate barcode sequences. Here we have developed an analytical framework, Selection of informative Amplicon Barcodes from Experimental Replicates (SABER), that identifies barcodes that are unique among a large set of experimental replicates. Amplicon barcodes were sequenced from the blood of 56 adult zebrafish divided into training and validation sets. Informative barcodes were identified and samples with a high fraction of informative barcodes were chosen by bootstrapping. There were 4.2 ± 1.8 barcoded HSC clones per sample in the training set and 3.5 ± 2.1 in the validation set (p = 0.3). SABER reproducibly quantifies functional HSCs and can accommodate a wide range of experimental group sizes. Future large-scale studies aiming to understand the mechanisms of HSC clonal evolution will benefit from this new approach to identifying informative amplicon barcodes.

HUGE pipeline to measure temporal genetic variation in Drosophila suzukii populations for genetic biocontrol applications

“…Other pipelines are limited to performing whole-sample variant calling without being able to determine any variant linkage ( 16 , 33 ). Other pipelines are also limited to sequencing tens or hundreds of individuals per sample ( 10 , 12 , 32 – 36 ), while we demonstrate that HUGE can be used on thousands of individuals per sample.…”

“…Other pipelines are limited to performing whole-sample variant calling without being able to determine any variant linkage (16,33). Other pipelines are also limited to sequencing tens or hundreds of individuals per sample (10,12,(32)(33)(34)(35)(36), while we demonstrate that HUGE can be used on thousands of individuals per sample. Due to its reliance on high-throughput amplicon sequencing, the HUGE pipeline has limitations when linking SNPs or haplotypes between different loci.…”

“…Preparing sequencing libraries using DNA from many independent samples at once has been generally termed Pool-seq ( 31 ). Previous studies have developed and utilized amplicon Pool-seq pipelines to discover rare SNPs and Cas9-induced gene edits ( 12 , 16 , 32 , 33 ). Most population genetics studies in SWD have also utilized amplicon sequencing approaches ( 34 – 36 ).…”

Microenvironmental control of hematopoietic stem cell fate via CXCL8 and protein kinase C

“…Barcoded zebrafish were grown to 3 months post fertilization (mpf), peripheral blood was sampled, GESTALT barcodes were sequenced, and the number of HSC clones contributing to peripheral blood was quantified using an unsupervised algorithm for selecting informative amplicon barcodes from experimental replicates (SABER). 30 Peripheral blood was serially resampled at 6, 9, 12, and 22 mpf. Accounting for repeated measures across all serial samples, animals in the sele:prkcda-2A-mCherry group had more HSC clones contributing to the peripheral blood compared to control clutchmates from 3 to 22 mpf ( Figure 2B ).…”

Hematopoietic stem cells and betaherpesvirus latency

“…Long-term transplantation experiments, in a diversity of species, suggest a clonal diversity model where the HSC compartment consists of a fixed number of different types of HSCs, each with a preprogrammed fate ( Muller-Siebrug et al., 2002 ; Lu et al., 2019 ; Teets et al., 2020 ). This coincides with the classic model of hematopoiesis where HS/HPCs maintain the stem cell compartment through regulated quiescence and self-renewal balanced with controlled apoptosis, proliferation, and differentiation as needed.…”