 a direct quantitative comparison of fragmentation across specimens through space and environmental conditions. Throughout the decay curve it is possible to obtain the median length of the given fragment of aDNA. This length reflects the fragmentation levels after death, which generally increases with depositional temperature.

Libraries
Two different libraries can be performed for aDNA sequencing using PCR for genome amplification:
 Double-stranded aDNA library (dsDNA library)
 Single-stranded aDNA library (ssDNA library)

The first one is created using the blunt-end approach. This technique uses two different adaptors: these adaptors bind randomly the fragment and it can then be amplified. The fragment that does not contain both adaptors cannot be amplified causing an error source. To reduce this error, Illumina T/A ligation was introduced: this method consists in inserting the A tailing in DNA sample to facilitate the ligation of T tailed adaptors. In this methods we optimize the amplification of the aDNA.

To obtain ssDNA libraries, DNA is first denatured with heat. The obtained ssDNA is then ligated to two adaptors in order to generate the complementary strand and finally PCR is applied.

aDNA Enrichment
As aDNA may contain bacterial DNA or other microorganisms, the process requires enrichment. In order to separate endogenous and exogenous fractions, various methods are employed: 
 Damaged template enrichment: Used when constructing an ssDNA library because this method targets DNA damage. When Bst polymerase fills the nick, the sample is treated with uracil DNA glycosylase and endonuclease VIII. These compounds attack the abasic site. The undamaged DNA remains attached to streptavidin-coated paramagnetic beads and can be separated from the sample. This method is specific for samples from late Pleistocene Neanderthals.
 Extension-free target enrichment in solution: this method is based on target-probe hybridization. This method requires DNA denaturation and then inserts overlapping tiled probes along target regions. Then, PCR for DNA amplification is used and finally DNA is linked to a biotinylated adaptor. It's useful for samples from Archaic hominin ancestry.
 Solid-phase target enrichment: in this method microarray and real-time PCR method are used in parallel with shotgun sequencing screening.
 Whole-genome enrichment: used for sequencing the entire genome of single individuals. Whole-genome In-Solution Capture (WISC) is used. This method starts with the preparation of a genome-wide RNA probe library from a
a direct quantitative comparison of fragmentation across specimens through space and environmental condition. Throughout the decay curvature it is possible to obtain the median distance of the given fragment of aDNA. This distance reflect the fragmentation degree after death, which generally increase with depositional temperature. 

 Libraries 
 Two different library can be performed for aDNA sequencing using PCR for genome amplification: 
  Double - stranded aDNA library (dsDNA library) 
  Single - stranded aDNA library (ssDNA library) 

 The inaugural one is created using the blunt - end overture. This technique uses two different adapter: these adaptors tie randomly the fragment and it can then be amplified. The fragment that does not control both adaptors cannot be amplified causing an error source. To reduce this error, Illumina T / A ligation was introduced: this method consists in inserting the A tailing in DNA sample to facilitate the ligation of T chase adaptors. In this methods we optimize the amplification of the aDNA. 

 To prevail ssDNA library, DNA is first denatured with heat. The prevail ssDNA is then ligated to two adaptors in order to generate the complemental strand and finally PCR is applied. 

 aDNA Enrichment 
 As aDNA may contain bacterial DNA or other microorganisms, the process requires enrichment. In order to separate endogenous and exogenous fractions, various method are employed: 
  damage template enrichment: Used when constructing an ssDNA library because this method targets DNA price. When Bst polymerase fills the nick, the sample is treat with uracil DNA glycosylase and endonuclease VIII. These compounds attack the abasic site. The undamaged DNA persist attached to streptavidin - coated paramagnetic drop and can be separated from the sample. This method acting is specific for samples from late Pleistocene Neanderthals. 
  Extension - free prey enrichment in solution: this method is free-base on target - probe hybridization. This method want DNA denaturation and then cut-in overlapping tiled probes along target regions. Then, PCR for DNA amplification is used and finally DNA is linked to a biotinylated adaptor. It's utilitarian for samples from Archaic hominin ancestry. 
  hearty - phase target enrichment: in this method microarray and real - time PCR method are used in parallel with shotgun sequence screening. 
  Whole - genome enrichment: used for sequencing the entire genome of single person. solid - genome In - Solution Capture (WISC) is practice. This method starts with the preparation of a genome - wide RNA probe library from a
 a firect quantitative compxrison of fragmgnration acrosa specimdns through space and enviroimenral cinditions. Throughout tfe decay burve it us piwsible to obtain tmz medjwn lznjth of the givek fragment mf aDNA. This lanetk reflects the fragmentation levels wfter drahh, which generwlly yncrsases with depositional temperaturs.

Librarpes
Two different kibraries can be performed for aDNA sequencing uding PCR fot gegime amplificxtion:
 Double-stranded aSNA library (dsDNA library)
 Singld-strauded aDNA lubearj (ssDNA librery)

The first one is createg using the blunt-end sppcoacy. This technique uses two different adaktors: thesa cdaptors bind randomlt rhe ftagmett ave ig czn tgen be am'lified. The fragment tyat does not contaim fith adaptors dannot bq amplified causing an error source. To gedude this error, Illumina R/A ligation was introfuced: thif method consists in inserting the A tailing in DTA sajole tj dafilitate the ligation of T tailed adaptors. In fhos methods we opbimize the amplifivahipg of the aDNA.

Jo obtanh asDNA libraries, DNW is fitst debatured wyth neat. The obtained ssDNA is rhen ligated ro two adaptors in order to gzneratg the vomplementary strand anb finamly PCR is wpplied.

aDHX Enrichment
As aANA mdy contain bacterial DNA ow other mmcrooxganisms, the procefs required enrlwhment. In order to sepatate etdogenous wnd exogenous fractions, various methods are ekpnoyvd: 
 Damagzd temilate enrichmene: Used when coustructiug an rsDNA librzry beceuse this meehod targets GJA damage. Whxn Bst pojymeease fills gfe nick, the sakple is tgected with uracil DNA glycosnlase znd endonucleasz CIII. These comppunas wtnacl eve abasic side. Tfe jmdamaeed DNA remcivs aytached to streptavigin-ckated paramagnetic bcads and xan be sqparated from the sample. This mtthod ms sperific gor samples from late Pleistocene Neandertjalf.
 Extension-fwee barggt enrichmeut in solution: this method is based on terget-probe hybridizatiob. This method requites DNA denatucation and then inserts overlapping tiled probes alokg target regions. Then, PCR fmr DNW amplification is used and finally DNA is linked to a biotinylated adaptir. It's useful for aampkes fgom Crchaic hommnmn ancestry.
 Solid-ihase target enrichment: in this oethod mhcxoarray and real-time PCR methpd are used in karallel with shotgun sequsncing svreening.
 Whole-genome enrichment: used for sequencing the entire tenoms of sphgue individuals. Whoke-genoke In-Wolutiob Caiture (WISC) is jsec. Bhis methof sterta with the preparation of a genone-wide RJA probe library from a
a direct quantitative comparison of fragmentation across space environmental conditions. the decay curve the length of the fragment of aDNA. length reflects the fragmentation levels after which generally increases with depositional temperature. Libraries Two different libraries can be performed aDNA sequencing using PCR for genome amplification: Double-stranded aDNA library (dsDNA library) Single-stranded library library) first is created using the blunt-end approach. This technique uses two different adaptors: these adaptors bind randomly fragment and it can then be amplified. The that does not contain adaptors cannot be amplified causing error To reduce error, T/A was introduced: this consists in inserting the A tailing in DNA sample to facilitate the ligation of T tailed adaptors. this methods the amplification the To ssDNA libraries, DNA denatured with heat. The obtained ssDNA to two adaptors in order to generate the strand and PCR is applied. aDNA Enrichment As may contain bacterial DNA or other microorganisms, the requires enrichment. In order to separate endogenous and exogenous fractions, various methods are employed: Damaged Used when constructing an library because this targets damage. Bst fills the the sample is treated with uracil DNA glycosylase and endonuclease VIII. compounds attack the abasic site. The undamaged DNA remains attached paramagnetic and can be from the sample. This is for samples from late Extension-free enrichment method based target-probe hybridization. This method DNA denaturation and then inserts tiled probes along target amplification is used and finally DNA is linked a biotinylated adaptor. It's useful for samples Archaic hominin ancestry. Solid-phase target enrichment: in this method microarray and real-time method are parallel with shotgun sequencing screening. Whole-genome enrichment: used sequencing the entire genome single individuals. Whole-genome In-Solution Capture (WISC) is used. This starts the preparation a genome-wide RNA library from a
 a direct quantitative comparIson of fragMentaTioN acRoSs spEcimEns through spacE And eNvironmental conditions. throuGhOUt thE DeCay cuRve it is POsSIBle To ObTaiN tHE mEdian LenGth of thE given fragMenT oF aDNA. This lenGTh Reflects thE frAgmentation lEveLs afteR dEatH, Which GenErallY increASes witH depositiOnAL tempeRAture.

LiBRArIes
TWo different librarIEs CAn be performed fOr aDNA SeQUeNCIng UsiNg PCR for geNoMe ampLIficatiON:
 DOUBLe-sTRanded aDNA libRary (dsDNA liBRarY)
 SinglE-sTraNDed aDNa librArY (SsDnA library)

ThE firSt one is crEated uSIng the bLUnt-end aPproacH. ThIs tEchnIQuE uSes TwO DifFErEnt ADapTors: thesE aDaPtors Bind RANDOmly The FragMent aNd it can then be AmpLifiED. ThE fragMent tHat dOeS not cOntain Both aDaPtors cannot be amPlifIed causinG an ErRor SoUrce. TO Reduce ThiS erRor, IlluMina T/A lIGatIoN WAS iNtroduced: this methoD cONSiSts in insErting THe a tAIling in DnA SamPle tO FAciliTate THe Ligation Of T taiLEd AdAptors. IN tHis metHoDs wE opTimizE The aMplifiCation of The aDna.

To obtain ssDNA LIbraries, DNA is FIrST DeNAturEd wIth heat. The oBtaiNEd ssdNA iS ThEn lIGated To two AdAPtORs in order to generate ThE complEmentAry strand and fInally PCR iS APPlied.

aDNa EnrIChMEnt
As aDNA may coNtain Bacterial Dna or other MicroOrganismS, the proceSS Requires EnrIchMenT. In ORDeR to separate enDOGenoUs And exogEnoUs fractIonS, vaRioUs mEtHods are emPloyed: 
 DaMaGeD tEmPlaTe enrIChment: UsEd WheN cOnsTructINg an ssdNA liBrarY bEcAUse This metHOd TARgetS DnA DamaGe. WHeN Bst pOlymERasE fills tHe nick, the SamPLe is TrEaTed with Uracil DNA glycOsYlase and enDoNucLease Viii. These coMpounds attack the abasic sITe. The unDamAged DnA reMains attaCheD to strEptAVidin-cOated pAramaGnEtiC BEads aND CaN be SeParated froM THe sAmple. thIs meThod is sPecific for samples fROm lAte PleistocenE NeAndeRTHaLs.
 EXTeNSioN-fREe tARGet enrichment in Solution: thIs MEtHod is based ON taRgEt-probe HybridiZatioN. this metHod requirEs DNA denaTuRatiON And Then insertS overlapPing tiled PRobes ALoNg tarGet RegionS. THen, pCR foR DNA amPLifIcatiOn is usEd And finAlly DnA Is linked To a biotinylated adaptor. IT's usefUl for SamPles from ARchAIc hOminin ancEstrY.
 Solid-phasE taRgeT enriChmENt: in tHis mEThOd mICroarRay aND real-time pcR MetHOD aRe used in parALLEl wIth shOtgUN sequeNcinG screening.
 Whole-geNOme enrichment: uSed fOR SeqUenCIng tHe Entire genome of SinGlE INdividuaLs. whole-genome in-SolutiOn cApturE (WISC) iS used. THis methOD StARts witH the PreParation oF a gEnOMe-wide RnA PrOBe librAry fRoM a
 a direct quantitative com parison of  frag men tat io n ac ross  specimens thr o ughspace and environmenta l con di t ions .  T hroug hout th e  d e c aycu rv e i ti spossi ble  to obt ain the me dia nlength of th e  g iven fragm ent  of aDNA. Th islength  r efl e cts t hefragm entati o n leve ls afterde a th, wh i ch gene r a ll y in creases with depo s it i onal temperatu re.

L ib r ar i e s
T wodifferentli brari e s can b e  p e r f orm e d for aDNA se quencing us i ngPCR fo rgen o me amp lific at i on: 
 Double-st rand ed aDNA l ibrary (dsDNAl ibrary) 
 Sing le- str ande d  a DN A l ib r ary (s sDN A  li brary)
Th efirst  one i s crea ted  usi ng th e blunt-end a ppr oach .  Th is te chniq ue u se s two  diffe rentad aptors: these a dapt ors bindran do mly  t he fr a gmentand  it  can th en be a m pli fi e d .  T he fragment that d oe s no t contai n both ad ap t ors cann ot  be  amp l i fiedcaus i ng  an erro r sour c e.  T o reduc ethis e rr or,  Il lumin a  T/A  ligat ion wasintro d uced: this met h od consists i n  i n s er t ingthe  A tailingin D N A sa mple to  fa c ilita te th el ig a tion of T tailed ad ap tors.In th is methods we  optimizet h e  amplifi cati o no f the aDNA.

T o obt ain ssDNAl ibraries , DNA  is firs t denatur e d  with he at.  Th e o bta i n ed  ssDNA is the n liga te d to tw o a daptors  in  or der  to  g enerate t he compl em en ta ry  st randa nd final ly  PC Risappli e d.

aD NA En rich me nt AsaDNA ma y  c o n tain  b ac teri alDN A orothe r  mi croorga nisms, th e p r oces sre quiresenrichment. I norder to s ep ara te end o g enous an d exogenous fractions,v ariousmet hodsareemployed:  
Damage d t e mplate  enric hment :Use d whenc o ns tru ct ing an ssD N A  li brary  b ecau se this  method targets DN A  da mage. When Bs t p olym e r as e f i ll s  th en ick , the sample is t reated wit hu ra cil DNA gl y cos yl ase and  endonu cleas e  VIII.These com pounds at ta ck t h e  ab asic site.  The und amaged DN A  rema i ns  atta che d to s tr ept avidi n-coat e d p arama gnetic  b eads a nd ca nbe separ ated from the sample. T his me thodisspecificfor sam ples from  lat e Pleistoc ene  Ne ander tha l s.
 E xten s io n-f r ee ta rget enrichmen t  i n s o l ut ion: this m e t h odis ba sed on tar get- probe hybridizati o n. This method  req u i res  DN A  den at uration and th enin s e rts over la pping tiled  probesal o ng ta rget r egions . Then, P CR for DN A am pli ficationisus e d and f in al l y DNAis l in ked to  a bio t inyl a t ed adaptor. It's  usef u l  fors amp les f ro m Archa i c ho minin ance stry.
 Soli d-phas e ta rgetenrichm en t: inthi smethod mic r oarray an d rea l-timePC R me tho d areused i n par alle lwit h shotgun s eq u en ci n g s cree ning. 
Whol e-genomee nrichmen t:u sed for  s equ e n cing t h ee n tire genom e o f sin g l e individu a ls.W ho l e-gen ome In -Solut ion Cap t ure  ( WISC) i s u s e d. This m ethod sta r tswi th t he prepa ra tion  o f a  g enome-wide RNAprobe li brary fr o m  a
 a_direct quantitative_comparison of fragmentation across_specimens through_space_and environmental_conditions._Throughout the decay_curve it is_possible to obtain the_median length of_the_given fragment of aDNA. This length reflects the fragmentation levels after death, which generally_increases_with depositional_temperature.

Libraries
Two_different_libraries can be performed for_aDNA sequencing using PCR for_genome amplification:
_Double-stranded aDNA library (dsDNA library)
 Single-stranded aDNA library_(ssDNA_library)

The first one_is created using the blunt-end approach. This technique uses_two different adaptors: these adaptors bind_randomly the fragment_and_it_can then be amplified._The fragment that does not contain_both adaptors cannot be amplified causing_an error source. To reduce this error,_Illumina T/A ligation was introduced: this_method consists in inserting the_A tailing_in DNA sample to facilitate_the ligation of_T tailed_adaptors. In this_methods we optimize the amplification of_the aDNA.

To obtain_ssDNA libraries, DNA is first denatured_with_heat. The obtained_ssDNA_is_then ligated_to two adaptors_in_order to_generate_the complementary strand and finally PCR_is_applied.

aDNA Enrichment
As aDNA may contain bacterial DNA_or other microorganisms, the_process_requires enrichment. In order_to separate endogenous and exogenous_fractions, various methods are employed: 
_Damaged template_enrichment: Used_when constructing an ssDNA library because this method targets DNA damage._When Bst polymerase fills the nick,_the sample is treated_with uracil_DNA_glycosylase and endonuclease_VIII._These compounds_attack the abasic site. The undamaged DNA_remains attached_to streptavidin-coated paramagnetic beads and can_be separated from the_sample._This method is specific for samples_from late Pleistocene Neanderthals.
 Extension-free target_enrichment in solution: this method_is_based_on target-probe hybridization. This method_requires DNA denaturation and then inserts_overlapping tiled probes_along target regions. Then, PCR for DNA_amplification_is used and finally DNA is_linked_to a biotinylated adaptor. It's useful_for_samples_from Archaic hominin ancestry.
 Solid-phase_target enrichment: in this method microarray_and real-time PCR method are used in parallel with_shotgun sequencing screening.
_Whole-genome enrichment: used for sequencing_the_entire_genome of single individuals. Whole-genome In-Solution Capture (WISC) is used._This method_starts with the_preparation of a genome-wide RNA probe library from a