lobSTR: a novel pipeline for short tandem repeats profiling in personal genomes

lobSTR: A Short Tandem Repeat

Profiler for Personal Genomes

Melissa GymrekRECOMB 2012

Why profile STRs?

Melissa Gymrek4/21/12 lobSTR

Huntington disease

Fragile X syndrome

Synpolydactyly

Medical Genetics

Intro. Algorithmic details Comparison Validation

(CAG)n

huntingtin

(GCA/GCG)n

HOXD13

(CGG)n

FMR15’

Why profile STRs?Forensics



Why profile STRs?

Despite multitude of applications, STRs are not routinely profiled in whole genome sequencing



Cell lineages

Challenges in profiling STRs from short reads

1. Only reads entirely spanning an STR are informative

Non-informative reads:

Informative reads:



Challenges in profiling STRs from short reads

2. Non-reference alleles present as large indels

3. PCR stutter noise complicates calling



lobSTR provides an end-to-end solution for STR-profiling

• Takes FASTA/FASTQ/BAM

• Reports read alignments and STR alleles

• Supports multi-threading





Our definition of STRs

• Motif size 2 through 6e.g. (AT)n, (CAG)n, (AGAT)n, (AGAGT)n, (AAAAAT)n

• At least 25bp long in the reference sequence

• Allow for imperfect repeat sequences (Tandem repeat finder score > 50)e.g. TATATACATATATATATATTATA

Aim: Find informative reads and characterize STR

SensingIntro. Algorithmic details Comparison Validation


Entropy score successfully detects STRs

Sj: sequence jΣ: symbol alphabet (dinucleotides)fi: frequency of symbol i

98.3%99.4%

AGCATATATATATATATATATATG

i fiAG 0.04GC 0.04CA 0.04AT 0.43TA 0.39TG 0.04… …



E(S) = 1.8

i fiA 0.46C 0.04G 0.08T 0.42

E = 1.54

CAGCTATTCGGGACTGAGCGGTAT

i fiA 0.21C 0.21G 0.33T 0.25

E = 1.97

i fiCA 0.04AG 0.08GC 0.08TA 0.08TT 0.04TC 0.04… …

E(S) = 3.71

Entropy score partitions reads

TTTTGTGTGAAACCATGCTCGAGTGTGTGTGTGTGTGTATGTGTGTGTGTGTGTGTGTGTGGTGTCTTAAGACTGAAATATCTAAGATTAACTTGG

Left flank Right flankSTR region

2/22/12



Repetitive sequences show distinct spectral signatures

ACCGT M =

1. Convert sequence to matrix 2. Compute power spectra

5mer4mer3merrandom

An STR of period k generates a strong signal in bin nc/k (c=0,±1,±2…)



The highest energy period gives the repeat unit length

• The period k whose first harmony has highest energy gives the repeat period

• Most frequent kmer gives the repeat unit


GTGTGTGTGTGTGTATGTGTGTGTGTGTGTGTGTG GT



Aim: Map STRs to the genome

AlignmentIntro. Algorithmic details Comparison Validation


We need to increase the specificity!

GT BWT

• Align only to known STR regions (Tandem Repeat Finder)

• Separate Burrows Wheeler Transforms store the left and right flanking regions of all genomic instances of each repeat

ATT BWT

GTGTGTGTGGTGTGTGTGGTGTGTGTGGTGTGTGTG

ATTATTATTATTATTATTATTATTATTATTATTATT



How to avoid gapped alignment?


GT BWT GT BWT

Divide and conquer approach:

A unique match anchors flanking regions to the genome and determines the repeat unit difference from reference

Locus 145 (-)Locus 172 (+)

Locus 172 (+)Locus 25,678 (+)

Locus 172

100bp

94bp

+6 bp



Aim: determine the STR alleles

AllelotypingIntro. Algorithmic details Comparison Validation


How to remove stutter noise?

• Use supervised learning to model PCR stutter noise

• Train on male sex chromosome STRs



Determine the most likely genotype

1. Enumerate possible allelotypes at each locus given observed reads R = 13,13,13,14,14,15 x GT

(13,13) (14,14) (15,15)(13,14) (13,15) (14,15)

3. Return maximum likelihood allelotype (13,14)


2. Calculate log likelihood for each allelotype (A,B) using the stutter model


lobSTR outperforms mainstream short read aligners at STR loci

lobSTR Bowtie BWA NovoAlign0

10000

20000

30000

40000

50000

60000R

eads

/ s

lobSTR Bowtie BWA NovoAlign0

10000

20000

30000

40000

50000

60000 speed#var reads483

0

222

293



lobSTR can detect pathogenic repeat expansions

Simulating a heterozygous carrier of HOXD13 pathology (7 trinucleotide expansion):

BWA fails to detect the pathogenic allele

lobSTR detects both alleles



lobSTR shows high concordance on biological replicates

BloodgDNA

SalivagDNA

Genotype consistency: how many loci give identical genotype callse.g. (10,10), (10,11) 0 (11,11), (11,11) 1

23456all

Period



lobSTR shows high concordance on biological replicates

BloodgDNA

SalivagDNA



Allele consistency: how many alleles agree between samplese.g. (10,10), (10,11) 0.5 (11,11), (11,11) 1

23456all

Period23456all

Period

Validation with DNA electrophoresis shows high accuracy

3 Runs of 101PE Illumina GAIIx

Genetic genealogy service

lobSTR

14 CODIS markers

gDNA

8 correct



4 partially covered (< 3x)

1 incorrect

1 not covered

Validation with DNA electrophoresis shows high accuracy


109bp PE reads, 1-2x77bp PE reads, 5-7xIllumina GAIIx

lobSTR

gDNA

HGDP samples

HGDP-CEPH panel~200 STR calls

At 3x coverage, lobSTR correctly returned 75% of genotypes calls and 85% of allele calls


Genome-wide STR profiling of 126x genome confirms known STR mutation trends

• 6.1 million reads aligned to 180,000 STR loci• 55% of called loci ≥20x had at least one non-reference allele

Het. ref/non-ref

Hom. non-ref

STR variation decreases with unit length

Hom. ref

Het. non-ref



Genome-wide STR profiling of 126x genome confirms known STR mutation trends

Longer STR regions show increased variation



lobSTR reports different variations for trinucleotide STRs in introns vs. exons



Introns 3 times as likely to have 1 non-ref allele

Introns 5 times as likely to have 2 non-ref alleles

0 frameshift mutations detected in exons vs. 1.9% in introns

Conclusion

• lobSTR is an end-to-end solution for STR profiling in personal genomes

• It outperforms mainstream aligners at STRs

• Validation by:- Consistency in biological replicates- Mendelian inheritance in trios- Capillary platform- Biological reasoning

• Thousands of STR variations in a single individualEnormous unexplored source of variation

lobSTR


jura.wi.mit.edu/erlich/lobSTR/

Acknowledgements

Funding:National Defense Science and Engineering Graduate Fellowship

Yaniv ErlichDavid Golan (Tel Aviv University)Saharon Rosset (Tel Aviv University)Dina EspositoMona Sheikh

Documents

lobSTR: a novel pipeline for short tandem repeats profiling in personal genomes