Approaches for in silicofinishing of microbial genome ... · Approaches for in silicofinishing of microbial genome sequences Frederico Schmitt Kremer1, Alan John Alexander McBride1

Approaches for in silico finishing of microbial genome sequences

Frederico Schmitt Kremer1, Alan John Alexander McBride1 and Luciano da Silva Pinto1

1Programa de Pós-Graduação em Biotecnologia (PPGB), Centro de Desenvolvimento Tecnológico,

Universidade Federal de Pelotas, Pelotas, Brazil.

Abstract

The introduction of next-generation sequencing (NGS) had a significant effect on the availability of genomic informa-tion, leading to an increase in the number of sequenced genomes from a large spectrum of organisms. Unfortunately,due to the limitations implied by the short-read sequencing platforms, most of these newly sequenced genomes re-mained as “drafts”, incomplete representations of the whole genetic content. The previous genome sequencing stud-ies indicated that finishing a genome sequenced by NGS, even bacteria, may require additional sequencing to fill thegaps, making the entire process very expensive. As such, several in silico approaches have been developed to opti-mize the genome assemblies and facilitate the finishing process. The present review aims to explore some free(open source, in many cases) tools that are available to facilitate genome finishing.

Keywords: microbial genetics, molecular microbiology, genomics, microbiology, draft genomes.

Received: September 25, 2016; Accepted: March 13, 2017.

Introduction

The advent of second generation of sequencing plat-forms, usually referred to as Next Generation Sequencing(NGS) technologies, promoted an expressive growth in theavailability of genomic data in public databases, mainlydue to the drastic reduction in the cost-per-base (Chain etal., 2009). Compared to the Sanger sequencing technique(Sanger et al., 1977), NGS platforms, like Illumina HiSeq,IonTorrent PGM, Roche 454 FLX, and ABI SOLiD, areable to generate a significantly higher throughput, resultingin a high sequencing coverage. However, they typicallyhave a lower accuracy (in terms of average Phred-score inthe raw data) and, in most cases, can only generate short-length reads (short-reads) (Liu et al., 2012). The term“short-read” is commonly used to refer to the data gener-ated by platforms such as Illumina, IonTorrent and SOLiD,as the length of their reads usually range from 30 bp (eg:SOLiD) to ~120 bp (e.g., Illumina HiSeq), which is smallerthan the length usually obtained by Sanger sequencing (~1kb), and by the PacBio and Oxford Nanopore platforms(commonly referred to as “long-reads”).

The higher throughtput obtained by NGS has stimu-lated the development of new algorithms and tools that arecapable of dealing with a larger volume of data and generat-ing genomic assemblies in a reasonable time. Traditionalsequence assemblers, like Phrap (http://www.phrap.org/

phredphrap/) and CAP3 (Huang and Madan, 1999), werereplaced with new ones, such as Velvet (Zerbino and Bir-ney, 2008), ABySS (Simpson et al., 2009), Ray (Boisvert etal., 2010), SPAdes (Bankevich et al., 2012) andSOAPdenovo (Luo et al., 2012), many of which were basedon the De Bruijn graph algorithm (Pevzner et al., 2001;Compeau et al., 2011). A large number of these softwareofferings are capable of assembling data from different se-quencing platforms, called “hybrid assembly”; however, allof them exhibit some limitations and, even after severalcorrections and optimization steps, the generation of a fin-ished genome continues to represent a complex task (Alkanet al., 2011).

Genome finishing is achieved by converting a set ofcontigs or scaffolds into complete sequences that representthe full genomic content of the organism without unknownregions (gaps) (Mardis et al., 2002), and aims a “closed”and full representation of the chromosome organization.On the contrary, a draft genome may be composed of a setof a few or several contigs or scaffolds (Land et al., 2015).Although useful for many studies, the unfinished and frag-mented nature of draft genomes may difficult analysis oncomparative genomics and structural genomics (Ricker etal., 2012). In addition, some genes may be missed if locatedin a region without coverage (e.g., edges of contigs/scaf-folds), or due to assembly errors (e.g., repetitive regionsthat are “collapsed” into a single one) (Klassen and Currie,2012).

Genome finishing represents a relevant step to reducedata loss and lead to a more accurate representation of thegenomics features of the organism of interest. The tradi-

Genetics and Molecular Biology, 40, 3, 553-576 (2017)Copyright © 2017, Sociedade Brasileira de Genética. Printed in BrazilDOI: http://dx.doi.org/10.1590/1678-4685-GMB-2016-0230

Send correspondence to Frederico Schmitt Kremer. Centro deDesenvolvimento Tecnológico, Universidade Federal de Pelotas,Campus Universitário, S/N, CEP 96160-000, Capão do Leão, RS,Brazil. E-mail: [email protected].

Review Article

tional way to close gapped genomes includes: (1) designprimers based on the edge of adjacent contigs, (2) PCR am-plification, (3) Sanger sequencing, and (4) local assembly,usually with (5) manual curation. As this process is verytime consuming, new in vitro approaches were developedto speed it up, including multiplex PCR (Tettelin et al.,1999), optical mapping (Latreille et al., 2007), and hybridsequencing and assembly (Ribeiro et al., 2012). This, how-ever, usually results in a drastic increase in the cost of thesequencing project. Therefore it is not surprising that fromthe 87,956 prokaryote genomes available in GenBank untilDecember 2016 (GenBank release 217), only a small frac-tion (~6,586) is finished (http://www.ncbi.nlm.nih.gov/genbank/).

In the last years, the availability of third-generationsequencing technologies, such as PacBio SMRT and Ox-ford Nanopore, has also provided another way to achievefinished genomes (Ribeiro et al., 2012). As these platformsusually generate reads with length > 10 kb, the assembly al-gorithms have to deal with less ambiguities and problem-atic regions (Koren and Phillippy, 2015). This makes thereconstruction of the chromosome sequences easier, butjust like second generation technologies, both platformshave their own limitations. In the earlier versions of thePacBio SMRT platform, for example, it used to present ahigh error-rate, so it was recommended to correct part ofthese errors using short-reads data (e.g., Illumina) (Koren etal., 2012; Au et al., 2012; Salmela and Rivals, 2014) beforeusing its result for any analysis. Although this problem hasbeen minimized with the improvements in the sequencingchemistry and base-calling process over the last years, thetime required for the sequencing, the cost of each run, andthe price of the equipment itself are still drawbacks, andthese are some of reasons as to why it is not more frequentlyapplied. In the case of Oxford Nanopore, there is a limitednumber of tools that can be used to pre-process and analyzeits data, and some types of errors (e.g., under-represen-tation of specific k-mers) are still recurrent (Deschamps etal., 2016). Therefore, second-generation platforms are stillthe most popular ones for a wide variety of applications,and remain as the cheaper alternative to obtain genomicdata.

Previous reviews that focused on genome assemblyand whole-genome sequencing analysis have already de-scribed some of the in silico tools that have the potential toimprove a genome assembly without the need for experi-mental data. Edwards and Holt (2013), Dark (2013) andVincent et al. (2016) have provided very comprehensivedescriptions of some of the main steps of data analysis inmicrobial genomes sequenced by next-generation technol-ogies, including de novo assembly, reference-based contigordering, genome annotation and comparative genomics,and these are useful starting points for those that are new tomicrobial genomics with NGS. Nagarajan et al. (2010)have also made some useful considerations regarding ge-

nome assembly and presented some methodologies for ge-nome finishing, but limited to a small set of approaches thatthey have developed to improve assemblies. Ramos et al.(2013) exemplified some assembly and post-assembly me-thods for genomes sequenced with IonTorrent platforms.Finally, Hunt et al. (2014) have also made a complete andaccurate benchmark analysis of different tools for assemblyscaffolding based on paired-end/mate-pair data. However,although very instructive, these reviews provide descrip-tions of some specific procedures, but do not give an in-depth view of the different finishing strategies, nor of thealgorithms that are used in background by different toolsfrom each category. Therefore, a more complete descrip-tion of these tools may be useful, especially for those re-searchers that are starting to work with microbial genomeanalysis and want to achieve better assemblies without re-lying on re-sequencing or manual gap-closing.

The present review aims at discussing some of thecategories of software that may be applied in the process ofassembly finishing, especially in the context of microbialgenome sequencing projects. A particular focus is placedon those that do not require additional experimental and/ornew sequencing data. The review intentionally focuses ontools that are freely available, at least for academic use, andomits proprietary software, like the CLC Genomics Work-bench (http://www.clcbio.com/), for example. This was notdue to anticipated differences in the performance or qualityof the results, but to adhere to the original intention: anoverview of tools that could be used by most researchers.As different approaches have been developed to improvegenomic assemblies, the description of these programs wasdivided into four main categories: scaffolding (placementof contigs into larger sequences by using experimental dataor information for closely-related genomes, and joiningthem by gap regions), assembly integration (generation of aconsensus assembly using multiple assemblies for the samegenome), gap closing (solving gaps by identifying theircorrect sequence), error correction (removal of misidenti-fied bases or misassembled regions) and assembly evalua-tion (quantification of the reliability of a genome assemblyand identification of its erroneous regions) (Table 1). Thesedifferent categories of programs can be combined, accord-ing to the type of data that is available (e.g., sequencingplatform and library that was used), and may help to reducethe fragmentation and improve the reliability of a genomeassembly. Based on the categories of software that are dis-cussed in the present review we have created the flowchartshowed in Figure 1, which may of help in choosing themost appropriate approach for genome finishing, depend-ing on the type of the data that is available. Examples of ge-nome projects that used some of the tools discussed in thepresent review are shown in Supplementary material (Ta-ble S1).

554 Kremer et al.

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e-ba

sed

scaf

fold

ing.

-N

otop

tim

ized

for

bact

eria

wit

htw

oor

mor

ere

plic

ons/

chro

mos

omes

.-

Res

ults

may

requ

ire

post

-pro

cess

ing

tore

cons

truc

tth

ese

-qu

ence

ofth

esc

affo

ld.

Java

:ht

tps:

//w

ww

.jav

a.co

m/

(Muñ

ozet

al.

,20

10)

Sup

plem

enta

ryda

taof

Muñ

ozet

al.

(201

0).

http

://d

x.do

i.or

g/10

.118

6/14

71-

2105

-11-

304

Sca

ffol

ding

SIS

-S

ingl

ere

fere

nce-

base

dsc

affo

ldin

g.-

All

ows

the

iden

tifi

cati

onof

geno

mic

inve

rsio

ns.

-N

otop

tim

ized

for

bact

eria

wit

htw

oor

mor

ere

plic

ons/

chro

mos

omes

.

MU

Mm

er(K

urtz

etal.

,200

4):

http

://m

umm

er.s

ourc

efor

ge.n

et/

(Dia

set

al.

,201

2)ht

tp:/

/mar

te.i

c.un

icam

p.br

:874

7.

Sca

ffol

ding

CA

R-

Sin

gle

refe

renc

e-ba

sed

scaf

fold

ing.

-A

llow

sth

eid

enti

fica

tion

ofge

nom

icin

vers

ions

and

tran

sloc

atio

ns.

-A

lso

avai

labl

eas

aw

ebse

rver

.-

Not

opti

miz

edfo

rba

cter

iaw

ith

two

orm

ore

repl

icon

s/ch

rom

osom

es.

MU

Mm

er(K

urtz

etal.

,200

4):

http

://m

umm

er.s

ourc

efor

ge.n

et/

PH

P:

http

s://

php.

net/

(Lu

etal.

,20

14)

http

://g

e-no

me.

cs.n

thu.

edu.

tw/C

AR

/

Sca

ffol

ding

RA

CA

-M

ulti

ple

refe

renc

e-ba

sed

scaf

fold

ing.

-O

ptim

ized

for

larg

ege

nom

esan

dw

ith

mul

tipl

ech

rom

o-so

mes

.-

Can

also

use

pair

ed-e

ndda

ta.

Non

e(K

imet

al.

,20

13):

http

://b

ioen

-com

pbio

.bio

en.i

lli-

nois

.edu

/RA

CA

/

Sca

ffol

ding

Rag

out

-M

ulti

ple

refe

renc

e-ba

sed

scaf

fold

ing.

-U

ses

phyl

ogen

etic

info

rmat

ion

toid

enti

fyth

em

ost

prob

a-bl

eor

ient

atio

nof

the

cont

igs

/sc

affo

lds.

Net

wor

kx(P

ytho

npa

ckag

e):

http

://n

etw

orkx

.git

hub.

io/

New

ick

(Pyt

hon

pack

age)

:ht

tp:/

/ww

w.d

aim

i.au

.dk/

~m

ailu

nd/n

ewic

k.ht

ml

Sib

elia

:ht

tp:/

/git

hub.

com

/bio

inf/

Sib

elia

(Kol

mog

orov

et

al.

,20

14)

http

s://

gith

ub.c

om/f

ende

rgla

ss/

Rag

out

Sca

ffol

ding

MeD

uSa

-M

ulti

ple

refe

renc

e-ba

sed

scaf

fold

ing.

-A

ccep

tsbo

thfi

nish

edan

ddr

aft

geno

mes

asre

fere

nce.

Bio

Pyt

hon

(Pyt

hon

pack

age)

:ht

tp:/

/bio

pyth

on.o

rg/

Java

:ht

tps:

//w

ww

.jav

a.co

m/

MU

Mm

er(K

urtz

etal.

,200

4):

http

://m

umm

er.s

ourc

efor

ge.n

et/

(Bos

iet

al.

,20

15)

http

s://

gith

ub.c

om/c

ombo

geno

mic

s/m

edus

a

Ass

embl

yin

-te

grat

ion

Min

imus

-C

anbe

easi

lyin

tegr

ated

wit

has

sem

bly

proj

ects

that

are

buil

ton

top

ofth

eA

MO

Spa

ckag

e.-

Req

uire

sex

peri

ence

wit

hth

eA

MO

Spa

ckag

ean

dit

sda

tafo

rmat

s.

AM

OS

pack

age

(Tre

ange

net

al.

,20

11):

http

://a

mos

.sou

rcef

orge

.net

/(S

omm

eret

al.

,20

07)

http

s://

sour

cefo

rge.

net/

pro-

ject

s/am

os/

Tab

le1

-co

ntin

ued

558 Kremer et al.

Cat

egor

yT

ool

Mai

nfe

atur

esD

epen

denc

es*

Ref

eren

ceD

ownl

oad

link

/w

ebse

rver

Ass

embl

yin

-te

grat

ion

Rec

onci

liat

or-

Cor

rect

sth

em

isas

sem

bled

regi

ons

ina

targ

etas

sem

bly

byco

mpa

ring

toan

alte

rnat

ive

asse

mbl

yfo

rth

esa

me

geno

me.

-Id

enti

fies

repe

titi

vere

gion

sth

atsu

ffer

edco

mpr

essi

ons

orex

pans

ions

.

MU

Mm

er(K

urtz

etal.

,20

04):

http

://m

umm

er.s

ourc

efor

ge.n

et/

(Zim

inet

al.

,20

08)

http

://w

ww

.gen

ome.

umd.

edu/

Ass

embl

yin

-te

grat

ion

MA

IA-

All

ows

the

inte

grat

ion

oftw

oor

mor

eas

sem

blie

s.-

Acc

epts

refe

renc

ege

nom

eto

perf

orm

scaf

fold

ing,

wha

tis

usef

ulfo

rth

ose

cont

igs

wit

hout

corr

espo

nden

cein

the

othe

ras

sem

blie

s.

Mat

lab:

http

s://

ww

w.m

athw

orks

.com

/M

UM

mer

:ht

tp:/

/mum

mer

.sou

rcef

orge

.net

/G

AIM

C(M

atla

bto

olbo

x):

http

://g

ithu

b.co

m/d

glei

ch/g

aim

c

(Nij

kam

pet

al.

,20

10)

http

://b

ioin

form

atic

s.tu

delf

t.nl

Ass

embl

yin

-te

grat

ion

CIS

A-

All

ows

the

inte

grat

ion

ofth

ree

orm

ore

asse

mbl

ies.

-C

orre

cts

mis

asse

mbl

edre

gion

san

dco

mpr

esse

d/

expa

nded

repe

ated

regi

ons.

BL

AS

T+

(Alt

schu

let

al.

,199

0;C

amac

hoet

al.

,20

09):

ftp:

//ft

p.nc

bi.n

lm.n

ih.g

ov/b

last

/M

UM

mer

(Kur

tzet

al.

,20

04):

http

://m

umm

er.s

ourc

efor

ge.n

et/

(Lin

and

Lia

o,20

13)

http

://s

b.nh

ri.o

rg.t

w/C

ISA

/

Ass

embl

yin

-te

grat

ion

GA

A-

Use

sth

eal

ignm

ent

betw

een

the

diff

eren

tco

ntig

sin

the

set

ofas

sem

blie

sto

gene

rate

anas

sem

bly

grap

h,w

hich

isex

-pl

ored

toid

enti

fyto

min

imal

set

ofin

depe

nden

tpa

ths.

BL

AT

(Ken

t,20

02):

http

s://

geno

me.

ucsc

.edu

/G

SM

appe

r:ht

tp:/

/454

.com

/

(Yao

etal.

,20

12)

http

://s

ourc

efor

ge.n

et/p

ro-

ject

s/ga

a-w

ugi/

Ass

embl

yin

-te

grat

ion

Mix

-G

ener

ate

anex

tens

ion

grap

hth

atre

pres

ents

the

conn

ecti

onbe

twee

nth

eco

ntig

s.-

Fil

ters

the

alig

nmen

tto

redu

ceth

eam

bigu

itie

sca

used

byre

peti

tive

sequ

ence

s.

Net

wor

kx(P

ytho

npa

ckag

e):

http

://n

etw

orkx

.lan

l.go

v/B

ioP

ytho

n(P

ytho

npa

ckag

e):

http

://b

iopy

thon

.org

/M

UM

mer

(Kur

tzet

al.

,20

04):

http

://m

umm

er.s

ourc

efor

ge.n

et/

(Sou

eida

net

al.

,20

13)

http

s://

gith

ub.c

om/c

bib/

MIX

Ass

embl

yin

-te

grat

ion

GA

M/

GA

M-N

GS

-R

equi

res

the

read

file

sto

perf

orm

the

asse

mbl

yin

tegr

atio

n.-

One

ofth

eas

sem

blie

sto

bem

erge

dis

defi

ned

as“m

as-

ter”

,whi

leth

eot

hers

are

defi

ned

as“s

lave

s”.

-A

llow

sth

eid

enti

fica

tion

ofm

isas

sem

bled

regi

ons

inth

em

aste

r,w

hich

are

corr

ecte

dbe

fore

the

gene

rati

onof

the

fina

las

sem

bly.

cmak

e:ht

tps:

//cm

ake.

org/

zlib

libr

ary:

http

://w

ww

.zli

b.ne

t/bo

ost

libr

arie

s:w

ww

.boo

st.o

rg/

spar

se-h

ash

libr

ary:

http

://g

oog-

spar

seha

sh.s

ourc

efor

ge.n

et/

(Cas

agra

nde

et

al.

,20

09;

Vic

edom

ini

etal.

,20

13)

http

s://

gith

ub.c

om/v

ice8

7/ga

m-

ngs

Ass

embl

yin

-te

grat

ion

Zor

ro-

Req

uire

sth

ere

adfi

les

tope

rfor

mth

eas

sem

bly

inte

grat

ion.

-R

emap

sth

ere

ads

back

toth

eco

ntig

san

did

enti

fies

mis

asse

mbl

edan

dre

peti

tive

regi

ons

base

don

the

cove

rage

.-

Spl

its

the

mis

asse

mbl

edco

ntig

san

dpe

rfor

ms

the

asse

mbl

yin

tegr

atio

nus

ing

Min

imus

.

AM

OS

(Tre

ange

net

al.

,20

11):

http

://a

mos

.sou

rcef

orge

.net

/B

ioP

erl

(Per

lm

odul

e):

http

://b

iope

rl.o

rgB

owti

e(L

angm

ead

etal.

,20

09):

http

://b

owti

e-bi

o.so

urce

forg

e.ne

t/M

UM

mer

(Kur

tzet

al.

,20

04):

http

://m

umm

er.s

ourc

efor

ge.n

et/

(Arg

ueso

etal.

,20

09)

http

://l

ge.i

bi.u

nica

mp.

br/z

orro

/

Tab

le1

-co

ntin

ued


Cat

egor

yT

ool

Mai

nfe

atur

esD

epen

denc

es*

Ref

eren

ceD

ownl

oad

link

/w

ebse

rver

Gap

clos

ing

Gap

Clo

ser

-G

ap-c

losi

ngfe

atur

eal

read

yin

tegr

ated

inth

eS

OA

Pde

novo

de

novo

asse

mbl

ypi

peli

ne-

Per

form

sa

loca

lre

asse

mbl

yin

the

gap

regi

onus

ing

the

read

slo

cate

din

the

edge

sof

the

surr

ound

ing

cont

igs.

Non

e(L

iet

al.

,20

10)

http

://s

oap.

geno

mic

s.or

g.cn

/

Gap

clos

ing

IMA

GE

-It

erat

ivel

ype

rfor

ms

are

map

ping

ofth

ere

ads

toth

eco

ntig

s,fo

llow

edby

the

sele

ctio

nof

thos

eth

atov

erla

pth

ega

pre

gion

and

alo

cal

reas

sem

bly.

Non

e(T

sai

etal.

,20

10)

http

s://

sour

cefo

rge.

net/

pro-

ject

s/im

age2

Gap

clos

ing

Gap

Fil

ler

-It

erat

ivel

ype

rfor

ms

are

map

ping

ofth

ere

ads

toth

eco

ntig

s,fo

llow

edby

the

sele

ctio

nof

thos

eth

atov

erla

pth

ega

pre

gion

and

alo

cal

reas

sem

bly.

-R

equi

res

info

rmat

ion

abou

tth

epa

ired

-end

libr

ary,

incl

ud-

ing

mea

nsi

zeof

the

inse

rt,i

tsst

anda

rdde

viat

ion

and

the

rel-

ativ

eor

ient

atio

nof

the

mat

es.

Non

e(B

oetz

eran

dP

irov

ano,

2012

)ht

tp:/

/ww

w.b

asec

lear

.com

/gen

om

ics/

bioi

nfor

mat

ics/

base

tool

s

Gap

clos

ing

Enl

y-

Iter

ativ

ely

perf

orm

sa

rem

appi

ngof

the

read

sto

the

cont

igs,

foll

owed

byth

ese

lect

ion

ofth

ose

that

over

lap

the

gap

regi

onan

da

loca

lre

asse

mbl

y.-

Ifa

refe

renc

ege

nom

eis

prov

ided

,ane

wsc

affo

ldin

gst

epca

nbe

perf

orm

edto

impr

ove

the

asse

mbl

y.

Bio

Pyt

hon

(Pyt

hon

pack

age)

:ht

tp:/

/bio

pyth

on.o

rg/

BL

AS

Tan

dB

LA

ST

+(A

ltsc

hul

etal.

,19

90;

Cam

acho

etal.

,200

9):

ftp:

//ft

p.nc

bi.n

lm.n

ih.g

ov/b

last

/C

dbfa

sta/

cdby

ank:

http

://c

ompb

io.d

fci.

harv

ard.

edu/

tgi/

soft

war

e/E

MB

OS

S:

http

://e

mbo

ss.s

ourc

efor

ge.n

et/

Min

imo

asse

mbl

er(T

rean

gen

etal.

,20

11):

http

://a

mos

.sou

rcef

orge

.net

/M

UM

mer

(Kur

tzet

al.

,20

04):

http

://m

umm

er.s

ourc

efor

ge.n

et/

Phr

ap:

http

://w

ww

.phr

ap.o

rg/p

hred

phra

pcon

sed.

htm

l

(Fon

diet

al.

,201

4)ht

tp:/

/enl

y.so

urce

forg

e.ne

t/

Gap

clos

ing

FG

AP

-U

ses

alte

rnat

ive

asse

mbl

ies

ofth

eta

rget

geno

me

toid

enti

fyre

gion

sth

atov

erla

pth

ega

p.M

atla

b:ht

tps:

//w

ww

.mat

hwor

ks.c

om/

(Pir

oet

al.

,20

14)

http

://w

ww

.bio

info

.ufp

r.br

/fga

p/

Gap

clos

ing

Sea

ler

-P

erfo

rms

alo

cal

re-a

ssem

bly

ofth

ega

pre

gion

sus

ing

dif-

fere

ntse

ttin

gsof

k-m

er,w

hat

may

help

inth

eso

lvin

gof

re-

gion

sw

ith

repe

titi

vese

quen

ces.

boos

tli

brar

ies:

ww

w.b

oost

.org

/sp

arse

-has

hli

brar

y:ht

tp:/

/goo

g-sp

arse

hash

.sou

rcef

orge

.net

/O

pen

MP

I:ht

tp:/

/ww

w.o

pen-

mpi

.org

(Pau

lino

etal.

,201

5)ht

tps:

//gi

thub

.com

/bcg

sc/a

byss

/tre

e/se

aler

-rel

ease

Tab

le1

-co

ntin

ued

560 Kremer et al.

Cat

egor

yT

ool

Mai

nfe

atur

esD

epen

denc

es*

Ref

eren

ceD

ownl

oad

link

/w

ebse

rver

Gap

clos

ing

GM

CL

oser

-M

ayus

ebo

thpa

ired

-end

read

san

dal

tern

ativ

eas

sem

blie

sto

perf

orm

the

gap-

clos

ing.

-A

ppli

esa

like

liho

odan

alys

isto

avoi

dth

eef

fect

ofm

isas

sem

blie

sin

the

alte

rnat

ive

asse

mbl

ies.

MU

Mm

er(K

urtz

etal.

2004

):ht

tp:/

/mum

mer

.sou

rcef

orge

.net

/B

LA

ST

+(A

ltsc

hul

etal.

,199

0;C

amac

hoet

al.

,200

9):

ftp:

//ft

p.nc

bi.n

lm.n

ih.g

ov/b

last

/B

owti

e(L

angm

ead

etal.

,200

9):

http

://b

owti

e-bi

o.so

urce

forg

e.ne

t/Y

AS

S(N

oéan

dK

uche

rov,

2005

):ht

tp:/

/bio

info

.lif

l.fr

/yas

s

(Kos

ugi

etal.

,20

15)

http

s://

sour

cefo

rge.

net/

pro-

ject

s/gm

clos

er/

Gap

clos

ing

Map

Rep

eat

-P

erfo

rms

are

fere

nce-

base

dsc

affo

ldin

gus

ing

acl

osel

y-re

late

dge

nom

epr

ovid

edby

the

user

.-

Use

sa

refe

renc

e-gu

ided

asse

mbl

yto

perf

orm

the

gap-

clos

ing

proc

ess.

BL

AS

T+

(Alt

schu

let

al.

,19

90;

Cam

acho

et

al.

,200

9):

ftp:

//ft

p.nc

bi.n

lm.n

ih.g

ov/b

last

/B

ioP

ytho

n(P

ytho

npa

ckag

e):

http

://b

iopy

thon

.org

/M

IRA

:ht

tp:/

/mir

a-as

sem

bler

.sou

rcef

orge

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Scaffolding

By definition, a contig consists of a contiguous se-

quence has no unknown regions or assembly gaps (but may

contain “N” that represent base-calling errors) (Staden,

1979). On the other hand, a scaffold consists of two or morecontigs that have been joined according to some linkage in-formation (e.g., paired-end reads, genome maps) (Huson etal., 2002). Paired-end or mate-pair libraries can be veryuseful in de novo genome assembly, and several tools usethe relative position information to connect contigs intoscaffolds (Hunt et al., 2014). In a similar way, with the in-crease in the availability of genomic sequences from a widevariety of species, other scaffolding alternatives were de-veloped to use one or multiple genomes as reference to or-der the contigs.

Paired-end scaffolding

Most of the de novo genome assemblers usually inte-grate scaffolding steps after the contig constructions, al-though it is also possible to use third-party tools aiming amore reliable result. The A5 assembly pipeline (Tritt et al.,2012), for example, uses de novo assembler IDBA (Peng etal., 2010) to construct the contigs and SSPACE (Boetzer etal., 2011) to generate scaffolds. The scaffolding withpaired-end reads usually consist of the alignment of readsto the contigs, followed by the identification of connectionsbetween different contigs using the relative-orientation in-formation and the estimated insert-size. ABySS (Simpsonet al., 2009), SOPRA (Dayarian et al., 2010), SOAPdenovo(Li et al., 2010), Bambus 2 (Koren et al., 2011), MIP(Salmela et al., 2011), Opera (Gao et al., 2011), SSPACE(Boetzer et al., 2011), SLIQ (Roy et al., 2012), SGA (Sim-pson and Durbin 2012), SCARPA (Donmez and Brudno2013), WiseScaffolder (Farrant et al., 2015) andScaffoldScaffolder (Bodily et al., 2016) are examples ofscaffolding tools based on paired-end information. Morerecently, the use of long-reads was also incorporated intoscaffolding tools such as AHA (Bashir et al., 2012) andSSPACE-LongRead (Boetzer and Pirovano, 2014).

ABySS (Simpson et al., 2009): The program abyss-scaffold, which comes with the ABySS assembly package(Simpson et al., 2009), uses the estimated mate-distancedistribution in paired-end reads to connect contigs and gen-erate scaffolds. The distance distribution can be calculatedby DistanceEst, that is also part of the package, and is alsoused by other assembly pipelines, such as SGA (Simpsonand Durbin, 2012). ABySS was developed to be used bothwith small and large genomes, and can be executed in acomputer clustering by using the Message Passing Inter-face (MPI), this being useful also in case of high-coveragedata and when dealing with multiple libraries. Like mostscaffolding programs, abyss-scaffold can be used also inthe scaffolding of contigs generated by third-party pro-grams. Finally, ABySS also supports scaffolding withlong-reads by using BWA-MEM for read alignment (Liand Durbin, 2009). The source code of the ABySS packageis available at the address http://www.bcgsc.ca/platform/bioinfo/software/abyss, and is developed to work on theLinux operating system.

562 Kremer et al.

Figure 1 - A flowchart demonstrating how and when the different genomefinishing approaches can be combined according to the data that is avail-able for the user. (a) Scaffolding using paired-end reads or long-reads,which is directly dependent on the way the genome was sequencing (plat-form, library), and sometimes performed as part of the de novo assemblyprocess. (b) Assembly integration, which consists in the combination ofdifferent de novo assemblies and generation of a consensus/extended as-sembly. Some programs use only the assemblies as input, while others usealso the sequencing reads. (c) The standard contig-ordering approachbased on a single reference genome, which consists in the identification ofsynteny blocks that guide the orientation of the contigs in the draft ge-nome, without taking into count the occurrence of genome inversionsother rearrangements. (d) The rearrangement-aware contig-ordering, thatidentifies potential sites of inversion and translocations based on signa-tures on the alignment against the reference genome. (e) The multi-ple-reference contig ordering, that may be more appropriate in those caseswhere there is no finished reference genome, but there is a relatively highnumber of close-related drafts, or when there are no apparent closest refer-ence to be used. (f) Assembly correction, which consists in the removing ofshort misassemblies, including base-substitutions and short insertions anddeletions. (g) Gap-closing, which consists in the joining of adjacentcontigs that used to be spaced by a gap. (h) Assembly evaluation, whichmay provide help to access the reliability of the assembly.

SOPRA (Dayarian et al., 2010): This scaffolding toolwas designed to improve assemblies generated by Velvet(Zerbino and Birney, 2008) and SSAKE (Warren et al.,2007), and targets the earlier sequencing platforms fromIllumina and ABI SOLiD. The program parses the read-placing file generated by these assemblers and extracts in-formation of paired-end/mate-pair reads, that is used to cal-culate the mean distance between pairs and the correctorientation. Based on this file, SOPRA also infers the con-nections between contigs by searches of those pairs of readswhere mates are in different contigs. The program is notfully automated, so each step of data processing must be ex-ecuted by a different script before the main scaffolding pro-cess. Another drawback is the limited support for differentde novo assemblers, as it requires read-placing files in AFGformat, and this is only produced by a few assemblers now-adays (e.g., Velvet, Ray and AMOS). SOPRA can be ob-tained from the website http://www.physics.rutgers.edu/~anirvans/SOPRA/.

Bambus 2 (Koren et al., 2011): It is part of the AMOSpackage (Treangen et al., 2011) and is both a genome andmetagenome scaffolding tool and an updated version of theSanger-based program Bambus targeting NGS data (Pop etal., 2004). The program requires read-placing informationto construct a contig-graph, and explore the graph to findconsistent connections between the contigs. As Bambus2can also be used to scaffold metagenomic assemblies, dif-ferent from other programs, it considers the effect of DNAsamples containing mixes of closely related organisms inthe assembly processes and reduces the chance of fragmen-tation and miss-joining by analyzing the molecular vari-ants. However, the use of Bambus 2 is not as simple as forother scaffolding tools, as it requires some experience withthe AMOS tools to generate its input file and processes theoutputs (Treangen et al., 2011). The AMOS package can beobtained from the SourceForge repository:https://sourceforge.net/projects/amos/.

MIP (Salmela et al., 2011): uses the concept of mixedinteger programming to generate a set of scaffolds from agenome assembly and a set paired-ends/mate-pair reads.First, readaligner (Mäkinen et al., 2010) is used to map theread-pairs back to the contigs. Then, the pairs are filtered toremove inconsistent connections, and the distances be-tween the contigs are estimated based on the mean distancebetween the mates, which is calculated for each library usedin the assembly. The connections in the generated scaffoldgraph have a minimum and maximum estimated length, de-rived from the library information. The MIP source code,along with usage instructions, is available athttps://www.cs.helsinki.fi/u/lmsalmel/mip-scaffolder/.

Opera (Gao et al., 2011): takes as inputs a collectionof contigs and mapped reads and generates a scaffold graphbased on the paired-end information. Frist, the program fil-ters the connections between contigs to remove possiblemiss-joining errors caused by chimeric pairs. The graph is

contracted, and the optimum orientation of the contigsinside the scaffolds is inferred by a dynamic programmingalgorithm that explores the search space. The algorithm canalso infer the occurrence of repeated genomic regions, usu-ally assembled into a single contig in case of short-reads. Inthis case, repeated regions are identified by comparing thecoverage of the contigs to the mean coverage of the wholegenome and selection those with value greater than 1.5times the genomic mean. The identification of these regionsallows a contig to be used in more than one scaffolds, whichcan provide a better assembly of repeated regions, but canalso result in misassemblies. Opera can be obtained from itsSourceForge repository https://sourceforge.net/projects/operasf/.

SGA (String Graph Assembler) (Simpson and Durbin2012): is a de novo genome assembler developed for thememory-efficient assembly of small and large genomes byapplying the method proposed by Myers (2005). As part ofits assembly pipeline, SGA also provide a scaffolding toolthat uses information from read alignment (in .BAM for-mat), that can be generated by a wide variety of mappingtools (Li et al., 2008; Langmead et al., 2009; Lunter andGoodson, 2011), and estimated distance between mates,generated by DistanceEst, from the ABySS package, toconnect contigs into scaffolds. SGA also supports scaffold-ing from multiple libraries, with different insert sizes, andwas optimized to work with Illumina data. SGA is availablefrom the GitHub repository https://github.com/jts/sga.

SCARPA (Donmez and Brudno, 2013): usespaired-end information to generate scaffolds, but takes intoaccount that not only chimeric reads may be to responsiblefor inconsistent linkages between contigs but also mi-sassembled sequences. It estimates the mean and standarddeviation of the distance between the mates, but only usesinformation from those contigs with length greater than theassembly N50. The connections between the contigs are es-timated based on the mate information and the calculatedmetrics, and if more than one library is provided, SCARPAprocess the scaffolding iteratively starting from the librarywith smaller insertion size. The program can be obtainedfrom the URL http://compbio.cs.toronto.edu/hapsembler/scarpa.html.

SSPACE (Boetzer et al., 2011) and SSPACE-LongRead (Boetzer and Pirovano, 2014): these scaffoldingprograms are currently distributed by BaseClear(http://www.baseclear.com), which also distributes thegap-closing program GapFiller (Boetzer and Pirovano,2012). SSPACE requires information about paired-end li-brary, including mean and standard deviation of distancebetween the mates and the expected orientation, whose val-ues can be predicted with the script “estimate_in-sert_size.pl”, distributed along with the program. The usermay choose between BWA (Li and Durbin, 2009) andBowtie (Langmead et al., 2009) for read mapping, the mini-mum number of connections to link two contigs, the num-


ber of bases that will be removed from the border of thecontigs (as they usually contain errors), and the number ofiterations. For SSPACE-LongRead, the target assembly isaligned to a collection of long-reads using BLASR (Chai-sson and Tesler, 2012) and the alignments are filtered andrefined to find the best orientation. Both SSPACE andSSPACE-LongRead can be requested from the BaseClearwebsite http://www.baseclear.com/genomics/bioinformatics/basetools/.

Hunt el al. (2014) have performed an extensive com-parison of the scaffolding tools and demonstrated that thequality of the resulting scaffolds is directly affected by theread-mapping program and the complexity of the genome.For the tested datasets, the best results were obtained bySGA, SOPRA and SSPACE, although all tested tools pre-sented a certain percentage of miss-joined scaffolds in theiroutputs. Some scaffolding tools (e.g., SGA) use pre-aligned reads as input, so the user is able to test and choosethe read mapper. In this case, it is important to try differentread mappers, taking into account that platform-specificbias and read quality may have a drastic effect on the qual-ity of the alignment (Hatem et al., 2013; Caboche et al.,2014). When using mate-pair libraries (long-insert paired-

end reads) it is also important to check if the scaffolder wasdesigned to support it, or if it was just designed for stan-dard, short-insert, paired-end reads. As mate-pairs maypresent a relatively high rate of “false mates”, special caremay be taken when working with this type of data.

Single reference-based scaffolding

In many cases the pairing information is not enoughto generate a reliable reconstruction of the genome’s struc-ture, or simply, the genome was not sequenced usingpaired-reads, but with single-end sequencing. In order toovercome this, some tools were developed to use a refer-ence genome as a template to perform the contig orderingand relative positioning (Figure 2). Software like MUMmer(Kurtz et al., 2004), ABACAS (Assefa et al., 2009),CONTIGuator (Galardini et al., 2011) and Mauve (Darlinget al., 2004; Rissman et al., 2009) are able to identify themost probable orientation of the contigs, but may generateincorrect results in the case of genome inversions of trans-locations. On the other hand, SIS (Dias et al., 2012), CAR(Lu et al., 2014), and FillScaffolds (Muñoz et al., 2010)consider the occurrence of changes in the genomic struc-ture and take these phenomena into account during the

564 Kremer et al.

Figure 2 - Reference-based contig ordering. (a) The program takes a set of contigs (or scaffolds) and (b) aligns these to a reference genome to identify themost probable relative orientation of the sequences in the draft genome. (c) Regions not covered by the contigs represent gaps and may be sequencing/as-sembling artifacts or natural deletions. Based on the relative position of each contig, a scaffold is created.

analysis, generating a more accurate reconstruction. Allthese tools use information from a single genome as refer-ence, however more recently, some tools, such as Ragout(Kolmogorov et al., 2014) and MeDuSa (Bosi et al., 2015),were developed to use information from multiple referencegenomes, allowing an evolutionary-based inference ofstructural re-arrangements. These multiple reference-basedcontig ordering tools will be discussed in the next section.

MUMmer (Kurtz et al., 2004): is a genome-scale se-quence alignment tool which can be applied to perform thealignment of a set of contigs/scaffolds to a reference ge-nome, allowing a wide variety of applications in genomicanalysis and NGS data processing, including reference-guided scaffolding. The two main algorithms of theMUMmer package are NUCmer, which performs a stan-dard DNA-DNA alignment, and PROmer, which performsan alignment of the six reading frames of both sequences(leading to a more sensitive result, especially in the case ofhighly divergent organisms). The package also includesother tools, such as delta-filter, that can be used to removethe ambiguities in the alignments and select those that aremore relevant for the analysis. Many scaffolding tools, likeABACAS (Assefa et al., 2009), CONTIGuator (Galardiniet al., 2011) and MeDuSa (Bosi et al., 2015), are built ontop of MUMmer and take advatange of its performance, butalso add new features to improve the output. MUMmer it-self does not provide the sequence of the scaffold, just thepositions of the alignments. Therefore, it is necessary toperform a post-processing of the results to obtain the se-quence of the scaffolds. MUMmer can be obtained from itsSourceForge repository http://mummer.sourceforge.net/.

ABACAS (Algorithm-based Automatic Contigua-tion of Assembled Sequences) (Assefa et al., 2009): can useNUCmer or PROmer from the MUMmer (Kurtz et al.,2004) package to align the contigs against a reference ge-nome. The regions that do not have an equivalent sequencein the contig set are filled with Ns, indicating gaps.ABACAS can also be used to design PCR primers to am-plify the unknown regions by integrating Primer3 (Ko-ressaar and Remm, 2007; Untergasser et al., 2012).ABACAS can be obtained from its SourceForge repositoryhttp://abacas.sourceforge.net/, and as part of the PAGITpackage (Swain et al., 2012), available at http://www.sang-er.ac.uk/science/tools/pagit.

CONTIGuator (Galardini et al., 2011): usesABACAS (Assefa et al., 2009) to perform the contig order-ing, but adds support to multiple references, which may beuseful in the case of organisms that have more than onechromosome. BLAST (Altschul et al., 1990; Camacho etal., 2009) is used to align the contigs used as input with thereference sequences to identify the correct reference foreach sequence. Then, ABACAS (Assefa et al., 2009) isused, and its results are integrated with the BLAST align-ment to generate a final assembly. CONTIGuator can beobtained from its SourceForge repository

http://contiguator.sourceforge.net/, and is also available asa webserver http://combo.dbe.unifi.it/contiguator.

Mauve (Darling et al., 2004; Rissman et al., 2009): isan alignment tool that can handle and align multiple ge-nomes and identify regions of high similarity called Lo-cally Collinear Blocks (LCBs). One of the program’s fea-tures, Mauve Contig Mover, performs contig orderingusing the same algorithm (Rissman et al., 2009). The pro-gram runs in an iterative mode, generating and optimizingthe contig orientations based on the reference until nochange is possible that can improve the model. A directoryis generated for each iteration that contains inputs to visual-ize the genome in Mauve and a FASTA file with the sortedcontigs. The Mauve aligner can be obtained from the URLhttp://darlinglab.org/mauve/mauve.html.

FillScaffolds (Muñoz et al., 2010): analyzes the ge-nomic distance between the contig set and a reference ge-nome and generates an ordered sequence throughidentifying orthologous genes. It considers the effects ofthe evolutionary distance in the case of missing genes, andthen uses the position of the orthologos present in the refer-ence to order the contigs. The source code of FillScaffoldsis available as a supplementary data of the Muñoz et al.(2010) paper at:http://bmcbioinformatics.biomedcentral.com/arti-cles/10.1186/1471-2105-11-304.

SIS (Scaffolds from Inversion Signatures) (Dias etal., 2012): takes as input a set of contigs in FASTA formatand a coordinate file generated by NUCmer or PROmer(Kurtz et al., 2004) after these contigs have been alignedwith the reference sequence. Using the coordinates, theprogram searches for inversion signatures and generates acollection of orientations of the sequences that can be usedto construct the scaffolds. The source code of SIS can beobtained from the URL http://marte.ic.unicamp.br:8747.

CAR (Contig Assembly using Rearrangements) (Luet al., 2014): uses NUCmer and PROmer in combination,unlike ABACAS (Assefa et al., 2009) and SIS (Dias et al.,2012), that use the result of only one. Based on the coordi-nates, CAR uses a block permutation model to generate thecontig order by considering not only the effect of the ge-nomic inversions, but also the occurrence of transpositions(Li et al., 2013). CAR can be used from the webserverhttp://genome.cs.nthu.edu.tw/CAR/, where the source codeis also available for download.

Considering the main algorithm of each program, it isimportant to keep in mind that the most appropriate tool fora given task will depend on the organism and the availabil-ity of the reference genomes. ABACAS (Assefa et al.,2009) is very useful if the reference genome is larger thanthe target genome (considering the sum of the length of allcontigs, and that all contigs have a homologous region inthe reference), and the primer designing tools might behelpful in some cases; however, its sensibility decreases incases of structural divergence. In such cases, other tools,


like CONTIGuator (Galardini et al., 2011) and Mauve(Darling et al., 2004; Rissman et al., 2009), may be moreeffective. Finally, SIS (Dias et al., 2012) and CAR (Lu etal., 2014) are indicated if the draft genome may presentgenomic inversions or transpositions. For most of the appli-cations, these tools usually provide reliable results, espe-cially for organisms that do not show a very variable ge-nomic organization, and/or when there are enough finishedgenomes to properly choose the best reference. However,in some situations it may be necessary to evaluate differenttools and references to check which one provides the bestresults. Finally, a single-reference may also lead to an“overfitted” ordering, especially when the reference issmaller, or in case of genomic inversions and translo-cations.

Multiple reference scaffolding

Sometimes it is very difficult to identify the most ap-propriate reference genome to use for contig ordering, es-pecially when structural rearrangements are commonevents in the genus/species of interests. Additionally, whenusing BLAST to identify the most “close-related” strainfrom a database of already finished genomes, it is not usualto find different strains as best hit for each contig. Finally,there are also those cases where no finished genome isavailable, but there are draft genomes of related strains. Inthese cases it would not be appropriate to use programs thattake into account alignments against only one reference, sodata from multiple organisms should be considered. Theuse of multi-references is relatively recent and another con-sequence of the advent of NGS, as there is more draftgenomes than finished ones available in public databases.Examples of algorithms and programs that use this ap-proach are RACA (Kim et al., 2013), Ragout (Kolmogorovet al., 2014) and MeDuSa (Bosi et al., 2015).

RACA (Reference-Assisted Chromosome Assem-bly) (Kim et al., 2013): uses local sequence alignment toidentify co-linear synteny blocks. The synteny blocks arefiltered using a length threshold, and based on the referencegenomes, the probability of each synteny block adjacent tothe others is calculated. This probability can also be com-bined with paired-end information to identify the mostprobable set of scaffold. The source code of RACA can beobtained from the URL http://bioen-compbio.bioen.illi-nois.edu/RACA/.

Ragout (Kolmogorov et al., 2014): uses phylogeneticinformation and synteny blocks to order a set of contigsfrom a target genome using multiple genome references.First, Sibelia (Minkin et al., 2013) is used to identifysynteny blocks shared by the target and the reference se-quences. Based on the synteny, the nucleotides of the ge-nomes are represented as sequences of blocks, and the best“block orientation” is identified by a maximum parsimony,taking into account the block order in the reference ge-nomes. The source code of Ragout can be obtained from its

repository at GitHub https://github.com/fenderglass/Ra-gout.

MeDuSa (Multi-Draft based Scaffolder) (Bosi et al.,2015): is a graph-based scaffolder that uses informationfrom multiples references, which can be finished or draftgenomes. The program uses NUCmer to alignment the tar-get genomes to the references and construct a weightedgraph based on the alignments were the nodes of the graphare connected by identifying those contigs that aligned tothe same sequence in the references. In the next step, theorientation of each contig is assigned based on the align-ment information and the most-probable ordered identifiedin the graph. The source code of MeDuSa can be obtainedfrom the repository at GitHub https://github.com/combogenomics/medusa.

Assembly integration

Different assemblers, or even the same assembler ex-ecuted with different configurations, may produce differentresults. Minimum coverage, coverage cut-offs, minimumcontig length, and k-mer size are examples of just some pa-rameters that can affect the decisions of the assembler dur-ing the construction of the contigs (Baker, 2012). The waylow-quality reads are treated, or how the correct paths in theassembly graph are constructed, is also different for eachprogram. As different assemblies may present differentrepresentations of a given region in the genome, the con-struction of a “consensus assembly” can be an effectivemethod of reducing assembly errors and generating an opti-mized set of contigs. This process, which is sometimescalled “assembly reconciliation,” “assembly merging,” or“assembly integration,” can receive as input only a set ofassemblies, as implemented in Minimus (Sommer et al.,2007), Reconciliator (Zimin et al., 2008), MAIA (Nijkampet al., 2010), CISA (Lin and Liao 2013), GAA (Yao et al.,2012) and Mix (Soueidan et al., 2013), or both a set of as-semblies and the reads used for the assembly, as is the casewith GAM-NGS (Vicedomini et al., 2013) and Zorro(Argueso et al., 2009).

Minimus (Sommer et al., 2007): is an assembly toolfrom the AMOS package (Treangen et al., 2011). Initiallyconceived to perform assembly of small genomes, it wasposteriorly adapted for assembly integration. The main al-gorithm is based on the overlap-layout-consensus paradigm(Peltola et al., 1984), which involves taking a set of se-quences and performing several alignments to identifyoverlaps. The information provided by the alignments isused to construct a graph that is minimized by a combina-tion of algorithms (Myers, 1995, 2005) to generate a finalassembly. Minimus is available as part of the AMOS pack-age, which can be obtained from the SourceForge reposi-tory https://sourceforge.net/projects/amos/.

Reconciliator (Zimin et al., 2008): uses NUCmer,from the MUMmer package (Kurtz et al., 2004), to identifyassembly errors by comparing a template with a secondary

566 Kremer et al.

assembly. With the alignments, the tool is able to identifythe regions that have possibly suffered compression or ex-pansion due to assembly errors in repetitive DNA se-quences. The source code of Reconciliator is available fromthe URL http://www.genome.umd.edu/.

MAIA (Multiple Assembly Integrator) (Nijkamp etal., 2010): uses the overlap-layout-consensus paradigm in asimilar way to Minimus to construct a graph based on theoverlaps identified by MUMmer (Kurtz et al., 2004). Theconnections in the graph are used to construct a new assem-bly, and contigs that have no connection can be integratedwith the assembly using a reference genome as a template.MAIA was implemented on top of the Matlab program-ming language and is available as a package for it that canbe obtained from the URL http://bioinformatics.tudelft.nl.

GAA (Graph Accordance Assembly) (Yao et al.,2012): is an assembly integration software that is based on ahomonymous data structure. Taking a set of contigs as in-put, the tool uses BLAT (Kent, 2002) to generate align-ments, identify overlaps and then generate a graph thatrepresents the connections between the contigs. GAA isavailable from the SourceForge repositoryhttp://sourceforge.net/projects/gaa-wugi/.

CISA (Contig Integrator for Sequence Assembly)(Lin and Liao, 2013): uses a four-step algorithm to generatethe merged assembly. First, a set of representative contigsis chosen from the individual assemblies. Assembly errorsare identified by aligning all sets to one another, and any re-gions that are present in only one sequence are consideredto be erroneous. In the event of errors, the contigs are bro-ken in the incorrect portion into smaller sequences. Thethird step consists of generating several alignments usingBLAST (Altschul et al., 1990; Camacho et al., 2009), andNUCmer (Kurtz et al., 2004) to identify the optimal lengthof repetitive sequences. The information generated in thethird step is used to construct the merged assembly in the fi-nal stage of the program. CISA can be obtained from theURL http://sb.nhri.org.tw/CISA/.

Mix (Soueidan et al., 2013): uses alignments gener-ated by NUCmer (Kurtz et al., 2004) to generate an exten-sion graph where the contigs are connected by theirborders. The alignments are filtered to remove repetitivesequences, and this information is used to generate a graph.Finally, the algorithm parses the graph to identify the Maxi-mal Independent Longest Path Set (MILPS) that representsthe final assembly. The Mix source code is available at theGitHub repository https://github.com/cbib/MIX.

GAM (Genomic Assemblies Merger) (Casagrande etal., 2009) and GAM-NGS (Genomic Assemblies Mergerfor Next Generation Sequencing) (Vicedomini et al.,2013): GAM takes an assembly as a template, which is re-ferred to as the “master”, and extends it using one or moresets of auxiliary assemblies (called “slaves”)

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