scRNA-Seq - Cell Ranger 定量

Cell Ranger 鏄?10X Genomics涓哄崟缁嗚優鍒嗘瀽涓撻棬鎵撻€犵殑鍒嗘瀽杞欢锛?strong>鐩存帴瀵?0X鐨勪笅鏈烘暟鎹繘琛屽熀鍥犵粍姣斿銆佸畾閲忋€佺敓鎴愬崟缁嗚優鐭╅樀銆佽仛绫讳互鍙婂叾浠栫殑鍒嗘瀽绛?/strong>銆傛墍浠ell Ranger鑳藉仛鐨勫垎鏋愭湁寰堝锛屾垜浠粖澶╀富瑕佸涓€涓婥ell Ranger鐨勫畨瑁呬互鍙婂鍗曠粏鑳濺NA-Seq鏁版嵁鐨勫畾閲忋€?/p>

Cell Ranger鐨勫畼缃戯細https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger

涓€銆佷笅杞戒笌瀹夎

1. 棣栧厛杩涘叆Cell Ranger瀹樼綉锛岀偣鍑诲涓嬫柟鐨凞ownload Link閾炬帴

濡傛灉鏄涓€娆¤繘鍏ヤ笅杞界晫闈紝闇€瑕佸～鍐欎竴浜涘熀鏈俊鎭紝濉啓瀹屽悗鐐瑰嚮continue鍗冲彲.

2. 鏍规嵁闇€姹備笅杞紺ell Ranger锛屽彲浣跨敤curl鎴栬€厀get鍛戒护涓嬭浇锛堝湪linux绯荤粺涓繍琛岄粦妗嗕腑鐨勫唴瀹瑰嵆鍙級;

wget -O cellranger-7.0.1.tar.gz "https://cf.10xgenomics.com/releases/cell-exp/cellranger-7.0.1.tar.gz?Expires=1669394099&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cHM6Ly9jZi4xMHhnZW5vbWljcy5jb20vcmVsZWFzZXMvY2VsbC1leHAvY2VsbHJhbmdlci03LjAuMS50YXIuZ3oiLCJDb25kaXRpb24iOnsiRGF0ZUxlc3NUaGFuIjp7IkFXUzpFcG9jaFRpbWUiOjE2NjkzOTQwOTl9fX1dfQ__&Signature=jE~gdVd5roKryS-0dA8R0RQazPC8ABzDNaUy40Opi9FrZqwUmkGCuNc5~Yn69BwGse81GTKIj5mMxZD0cnwu-A6oCbTFktns-WMGV6GaMa4sOVlHORcvwLjmF5lsb3tbFquwNNvxZe~NuBhUhFAZEBk7tRZjl4d~PqCYSDeRwmM~oR6mKUHRZ1yaKefxgy6R4CwQ~YaTcl7az5-Ucb0gmLkStYHQUuGmpy6M4LOds2kvcKcsDYMAw7Crghaja2Pn3rE-D71JyfHaBQ7EoM8gAijWumF1mYLK8yYCd1Tpb0JjWEzHR49eGX0iCQTZq~bfAcinhRLn90xZD2xfPlxhAA__&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA"

娉細榛樿涓嬭浇鏈€鏂扮増鐨凜ell Ranger锛屽鏋滈渶瑕侀€夋嫨涔嬪墠鐨勭増鏈彲鐐瑰嚮鍙充笅鏂圭殑绾㈡锛岄€夋嫨鎯宠鐨勭増鏈紙濡備笅鍥撅級锛?/p>

3. 瀹夎鍖呬笅杞藉畬涔嬪悗鐩存帴浣跨敤tar鍛戒护杩涜瑙ｅ帇鍗冲彲

tar -xzvf xxx.tar.gz

杩欐牱灏卞畬鎴怌ell Ranger鐨勫畨瑁呭暒

浜屻€佷娇鐢–ell Ranger杩涜鍗曠粏鑳炶浆褰曠粍娴嬪簭鏁版嵁锛坰cRNA-Seq锛夌殑瀹氶噺

cellrange count 鏄?cellrange 涓竴涓緢閲嶈鐨勫懡浠わ紝鐢ㄦ潵瀵瑰崟缁嗚優杞綍缁勬暟鎹繘琛屽熀鍥犵粍姣斿锛岀粏鑳炲畾閲忔渶缁堝緱鍒扮敤浜庝笅娓稿垎鏋愮殑鍗曠粏鑳炶〃杈剧煩闃碉紙榛樿鎯呭喌涔熶細瀵硅〃杈剧煩闃佃繘琛岃仛绫伙級銆?/p>

鍦ㄥ仛瀹氶噺涔嬪墠锛屾垜浠鍏堥渶瑕佸噯澶?缁勬枃浠讹細鍘熷fq鏂囦欢浠ュ強鐗╃鐨凴eferences锛堝叾涓寘鎷弬鑰冨熀鍥犵粍搴忓垪銆乬tf鏂囦欢浠ュ強star鐨勭储寮曟枃浠讹級銆?/p>

1. 鍘熷fq鏂囦欢

cellranger鐨勮緭鍏ユ枃浠舵牸寮忔槸fq鏍煎紡锛屽苟涓旀枃浠剁殑鍛藉悕涔熸槸鏈夎姹傦紝鏂囦欢鍛藉悕鏍煎紡濡備笅锛?/p>

[Sample Name]S1_L00[Lane Number][Read Type]_001.fastq.gz

It is highly likely that these files were initially processed with bcl2fastq. Once you track the origin of the file, you will rename the files in the following format:

[Sample Name]S1_L00[Lane Number][Read Type]_001.fastq.gz

Where Read Type is one of:

• I1: Dual index i7 read (optional)
• I2: Dual index i5 read (optional)
• R1: Read 1
• R2: Read 2

閾炬帴锛歨ttps://support.10xgenomics.com/single-cell-multiome-atac-gex/software/pipelines/latest/using/fastq-input#gex_rightname

濡傛灉fq鐨勬枃浠跺悕鏍煎紡涓嶅锛屽湪杩愯鐨勮繃绋嬩腑浼氬嚭鐜伴敊璇紝鎵€浠ユ渶寮€濮嬮渶瑕佺‘瀹氭枃浠跺悕鐨勬牸寮忎互鍙婅繘琛屼慨鏀广€備範鎯槸閲嶆柊鍒涘缓涓€涓洰褰曞苟涓旂敤杞繛鎺?/strong>灏嗗師濮嬫枃浠堕摼鎺ュ埌鏂扮殑鐩綍涓紝杩欐牱鍋氱殑濂藉鏄鍏堜笉浼氭敼鍙樺師濮嬫枃浠剁殑鍚嶅瓧锛堝鎬曚慨鏀逛簡鏂囦欢鍚嶅悗鏈変簺鏂囦欢娌℃湁鍚屾锛屽鑷存渶鍚庢壘涓嶅埌鍏蜂綋鐨勬枃浠讹級锛屽叾瀹炰篃涓嶄細鍗犵敤寰堝瀛樺偍锛堟瘯绔熸垜浠繕瑕佸湪澶圭紳涓敓瀛橈級锛屼笅闈㈠氨鏄垜浣跨敤鐨勯鏍硷細

2. 鐗╃鐨凴eferences

绗簩涓渶瑕佸噯澶囩殑鏂囦欢灏辨槸鐗╃鐨凴eferences銆?/p>

濂芥秷鎭氨鏄疌ell Ranger瀹樼綉宸茬粡涓烘垜浠彁渚涗簡浜哄拰灏忛紶鐨凴eferences锛屽鏋滃ぇ瀹剁殑鏍锋湰鏄汉鎴栬€呭皬榧犵殑鏌愪簺缁嗚優鍙互鐩存帴鍘籆ell Ranger瀹樼綉杩涜涓嬭浇銆?/p>

涓嬭浇娴佺▼鍜孋ell Ranger杞欢涓嬭浇娴佺▼涓€鑷达紝鍏朵腑涔熸槸鏈夊緢澶氱増鏈殑References鍙緵澶у閫夋嫨锛屼笅杞藉悗瑙ｅ帇灏卞彲鐢ㄤ簡锛?/p>

涓嬭浇缃戦〉锛歨ttps://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest

閭ｄ箞闂鏉ヤ簡锛屽鏋滄垜鐮旂┒鐨勬槸鍏朵粬鐗╃锛岄偅鎬庝箞鏋勫缓杩欎釜References锛?/strong>

cellranger鐨刴kref灏辨槸杩欎箞涓€涓姛鑳斤紝鍙敤瀵瑰叾浠栫殑鐗╃鏋勫缓cellranger闇€瑕佺殑References鏍煎紡锛屽彧闇€瑕佸噯澶?strong>鐗╃鐨勫弬鑰冨熀鍥犵粍搴忓垪鍜?strong>gtf娉ㄩ噴鏂囦欢灏卞彲浠ョ洿鎺ヨ繍琛屻€?/p>

杩欓噷灏变互鎷熷崡鑺ヤ负渚嬪瓙鏋勫缓References銆?br> 棣栧厛鍒涘缓瀛樻斁References鐨勭洰褰曪紝杩欐槸鎴戠殑涓€涓範鎯紝涔熸帹鑽愬ぇ瀹跺湪杩愯涓嶅悓姝ラ鐨勬椂鍊欒兘澶熷垱寤轰笓闂ㄧ殑鏂囦欢锛岃繖鏍蜂篃渚夸簬鏂囨。绠＄悊銆?/p>

mkdir refdata-cellranger-Arabidopsis-TAIR10

鍏蜂綋鍛戒护濡備笅锛?/p>

cellranger mkref \
--genome=TAIR10 \
--nthreads=10 \
--fasta=TAIR10.fa \
--genes=TAIR10_GFF3_genes.miRBase20.gtf

--genome锛氱敓鎴愮储寮曠殑鐩綍
--fasta锛氬熀鍥犵粍搴忓垪
--genes锛氬熀鍥犳敞閲婃枃浠讹紙gtf鏍煎紡锛?/p>

杩愯瀹屼笂闈㈢殑鍛戒护灏辨瀯寤哄畬绱㈠紩鍟

杩欓噷杩樿鎺ㄨ崘涓€涓繍琛岃剼鏈殑鍛戒护锛屽笇鏈涜兘澶熷澶у鏈夊府鍔┿€傛垜浠彲浠ヤ娇鐢?vi 缂栬緫鍣紝灏嗕笂闈㈢殑鍐呭瀛樻斁鍦ㄤ竴涓?shell 鑴氭湰涓紝鐒跺悗浣跨敤鍚庨潰杩愯 shell 鑴氭湰锛岃繖鏍峰悗鍙板湪杩愯鐨勫悓鏃讹紝鎴戜滑浠嶇劧鍙互鍦ㄥ綋鍓嶇晫闈㈣繘琛屽叾浠栨搷浣滐紝骞朵笖缃戠粶涓嶇ǔ瀹氱殑鏃跺€欎篃涓嶄細褰卞搷鎴戜滑鐨勮繍琛岋紝鎵€浠ラ潪甯告帹鑽愩€傦紙鑴氭湰鍚嶏細index_test.sh锛墌

鎶曞悗鍙扮殑鍛戒护鏄細

nohup sh index_test.sh >index_test.sh.o 2>index_test.sh.e &

杩欐牱鐨勮瘽涓棿鐨勮緭鍑烘枃浠朵細淇濆瓨鍦╥ndex_test.sh.o锛屽鏋滆剼鏈姤閿欏氨浼氫繚瀛樺湪index_test.sh.e涓€傛垜浠彲浠ラ€氳繃鏌ョ湅杩欎袱涓枃浠朵簡瑙ｈ繍琛岀殑杩涘睍銆傚彲浠ラ€氳繃浣跨敤jobs鍛戒护鏌ョ湅鍚庡彴杩愯鐨勫懡浠ゆ槸涓嶆槸杩樺湪銆?/p>

References鏋勫缓瀹屽悗灏变細鐢熸垚TAIR10鐩綍锛屽苟涓旇鐩綍涓嬬殑鏂囦欢鏈夛細

3. 瀹氶噺

鍦ㄦ墍鏈夋枃浠堕兘鍑嗗濂戒簡浠ュ悗锛屽氨鍙互浣跨敤count瀵瑰崟缁嗚優杞綍缁勬暟鎹繘琛屽畾閲忓暒銆?/p>

鍏蜂綋鍛戒护濡備笅锛堜竴鑸娇鐢ㄩ粯璁ゅ弬鏁帮級锛?/p>

cellranger count \
--id=sample_test  \
--transcriptome=/xx/ AT  \
--fastqs=/xxx/fastq_path  \
--localcores=8  \
--localmem=64

鍙傛暟瑙ｉ噴锛?/p>

• id锛氭牱鏈悕锛堝敮涓€鎬э級
• transcriptome锛氫笂涓€姝ュ垱寤虹殑绱㈠紩鐨?strong>鐩綍鍚?/strong>
• fastqs锛氫笅鏈烘暟鎹殑鐩綍鍚?/strong>
• localcores锛氬唴鏍?/li>
• localmem锛氬唴瀛?/li>

涓嬮潰鏄垜鐨勮剼鏈紝鍜屼笂闈㈡槸鍚屾牱鐨勮剼鏈牸寮弤

鎴愬姛杩愯涔嬪悗浼氱敓鎴恠ample_test鐩綍锛堣剼鏈腑id鍙傛暟鍚庨潰杈撳叆鐨勫唴瀹癸級锛屾渶缁堢粨鏋滈兘淇濆瓨鍦╯ample_test/outs涓€?/p>

鐩綍

• analysis锛歝ellranger鑱氱被鐨勭粨鏋?/li>
• filtered_feature_bc_matrix锛氳繃婊ゅ悗鐨勫崟缁嗚優琛ㄨ揪鐭╅樀锛堝悗缁彲浠ュ鎺ュ埌seurat涓級
• raw_feature_bc_matrix锛氳繃婊ゅ墠鐨勫崟缁嗚優琛ㄨ揪鏁版嵁锛堜竴鑸笉鎬庝箞浣跨敤锛?/li>

鏂囦欢

• possorted_genome_bam.bam锛氬崟缁嗚優姣斿鐨刡am鏂囦欢锛屽叾涓寘鍚簡姣忎釜reads鐨勪俊鎭?/li>
• web_summary.html锛氭姤鍛婄綉椤碉紙鍗曠粏鑳炲畾閲忓悗鐨勬姤鍛婏紝鍖呮嫭妫€娴嬪埌鐨勭粏鑳炴暟銆佸熀鍥犳暟銆乁MI銆佸垎缇ょ瓑绛夛級

END Cell Ranger

浠ヤ笂灏辨槸cellranger鐨勪笅杞姐€佸畨瑁呬互鍙婂垵姝ョ殑浣跨敤娴佺▼锛屽笇鏈涜兘澶熷府鍒板ぇ瀹跺暒~

涓夈€佺粨鏋滆В璇?/h3>

棣栧厛鎴戜滑浜嗚В涓€涓嬭繍琛屽畬Cell Ranger涔嬪悗锛屽湪鍝噷鍙互鐪嬪埌鐢熸垚鐨勭粨鏋?/p>

杩樿寰楁垜浠湪杩愯Cell Ranger鐨勬椂鍊欐湁涓弬鏁?-id鍚楋紵--id=XXX锛岃繖閲岀殑XXX灏辨槸鏈€缁堢敓鎴愮殑鐩綍锛岃鐩綍涓繚瀛樹簡杩愯杩囩▼涓墍鏈夌殑涓棿鏂囦欢銆佹棩璁版枃浠朵互鍙婃渶缁堢殑缁撴灉銆傚涓嬪浘锛?/p>

鍏朵腑outs鐩綍涓嵆淇濆瓨鐨勬渶缁堢粨鏋滐紝涔熸槸鎴戜滑鏈€鍚庨渶瑕佺殑銆傚綋鐒跺鏋滀腑闂村嚭鐜颁簡鎶ラ敊锛屾垜浠篃鍙互閫氳繃鏌ョ湅鏃ュ織鏂囦欢锛屼緥濡傦細_log锛屾煡鐪嬪叿浣撶殑鎶ラ敊鍘熷洜锛岄殢鍚庤繘琛屼慨鏀瑰嵆鍙€?/p>

02 缁撴灉鐩綍 outs

棣栧厛鎴戜滑鐪嬩竴涓媜uts鐩綍涓嬬殑鏂囦欢缁撴瀯锛屽涓嬪浘锛?/p>

杩欎簺缁撴灉涓富瑕佸垎鎴愪簡涓ら儴鍒嗭細
1. 闆嗙兢涓彲浠ヤ娇鐢ㄧ殑缁撴灉锛堝叿浣撶殑鍐呭鍙互鍙傝€冧笂鏈熸枃绔犫€?鍗曠粏鑳炲垎鏋愭祦绋嬩箣Cell Ranger 鈥?锛夛紱
2. 缃戦〉鐗堟姤鍛娿€?/p>

鏈湡鐨勯噸鐐规槸瑙ｈ缃戦〉鎶ュ憡涓殑鍐呭銆?/strong>

03 缃戦〉鎶ュ憡 web_summary.html

涓轰簡蹇€熶簡瑙ｅ拰鏂逛究鐨勪簡瑙ell Ranger瀹氶噺涔嬪悗鐨勭粨鏋滐紝鎴戜滑棣栧厛浼氭煡鐪媓tml鏂囦欢锛屽嵆 web_summary.html锛屼簡瑙ｅ垵姝ユ儏鍐点€傚涓嬪浘锛?/p>

鍙互鐪嬪埌璇ョ綉椤典腑涓昏鍒嗘垚浜嗕袱閮ㄥ垎锛歋ummary鍜孉nalysis.

04 Summary

1. 寮傚父缁撴灉璀﹀憡

濡傛灉鏁版嵁涓瓨鍦ㄥ紓甯告儏鍐碉紝缃戦〉鐨勪笂闈細鍑虹幇榛勮壊鐨勮鍛婁俊鎭€傛壘浜嗕竴涓嬩箣鍓嶉亣鍒拌鍛婁俊鎭紝濡備笅鍥撅細

褰撻亣鍒拌繖绉嶆姤閿欐儏鍐电殑鏃跺€欐垜浠笉瑕佹厡锛岄鍏堢湅涓€涓嬫槸鍝簺鍊煎紓甯革紝瀵规暟鎹湁鏃犲奖鍝嶄互鍙婅В鍐冲姙娉曘€傚湪Detail閮ㄥ垎浼氳缁嗚В閲婅繖涓弬鏁版槸浠€涔堬紝浠ュ強瑙ｅ喅鍔炴硶銆備緥濡備笂鍥句腑璇村埌鍦ㄨ繍琛孋ell Ranger鐨勬椂鍊欏彲浠ヨ皟鐢?-force-cells鍙傛暟锛岃繖涓弬鏁扮殑淇敼闇€瑕佷笉鏂殑灏濊瘯锛屾墍浠ヤ篃娌℃湁鍥哄畾鐨勫€拣煒?/p>

褰撶劧濡傛灉杩欎簺鎶ラ敊淇℃伅骞朵笉褰卞搷缁撴灉锛屾垜浠槸鍙互鐢ㄨ繖涓粨鏋滅户缁線鍚庡垎鏋愮殑锝?/p>

2. 缁嗚優鍜屽熀鍥犳暟鐨勭粺璁?/strong>

闅忓悗灏辨槸鏌ョ湅杩欐鍒嗘瀽涓崟鑾峰埌鐨勭粏鑳炴暟浠ュ強鍩哄洜鏁扮殑鎯呭喌锛屼粠杩欓噷灏辫兘澶ф鐭ラ亾鏁版嵁鐨勬儏鍐点€?/p>

鎴戜篃鍋氳繃濂藉10X鐨勬暟鎹紝涓€鑸崟鑾风殑缁嗚優鏁伴兘鏄?,000-10,000锛屽钩鍧囩殑鍩哄洜鏁板ぇ姒傛槸1,200-15,00锛屽ぇ瀹跺彲浠ョ湅鐪嬭嚜宸辩殑鏁版嵁鏄惁涔熷湪杩欎簺鑼冨洿鍐呫€傚鏋滆繖浜涘€奸兘鏄湪鍙帴鍙楃殑鑼冨洿锛岄偅涔堝氨鍙互杩涘叆涓嬩竴姝ョ殑鍒嗘瀽鍟

3. 缁嗚優鐨勯€夊彇

闅忓悗灏辨槸缁嗚優鐨勯€夊彇浜嗭紙涔熸槸涓€涓浉褰撻噸瑕佺殑鍥撅級锛屽府鍔╂垜浠洿鍔犵洿瑙傜殑绛涢€夌粏鑳烇紙濡備笅鍥撅級

鍏堟垜浠厛鏉ョ湅涓€涓嬩笂鏂圭殑鎶樼嚎鍥炬€庝箞鐪嬶細

Y杞存槸姣忎釜缁嗚優涓璘MI鐨勫€硷紝X杞存槸鍗曚釜缁嗚優鐨勬寜鐓MI澶у皬鐨勬帓搴忥紙闄嶅簭锛夛紝鎵€浠ヨ繖涓浘涓殑鏇茬嚎鏄笅闄嶇殑瓒嬪娍銆傝摑鑹茬殑绾挎槸閫夊彇鐨勭粏鑳烇紙鍜?strong>2. 缁嗚優鍜屽熀鍥犳暟鐨勭粺璁?/strong>涓殑缁嗚優鏁版槸涓€鑷寸殑锛夛紝鐏拌壊鐨勭嚎鏄儗鏅€?/p>

姝ｅ父鐨勬暟鎹潵璇翠細鏈変袱涓笅闄嶇殑瓒嬪娍锛堝涓嬪浘锛夛紝绗?涓笅闄嶇殑瓒嬪娍锛氬尯鍒嗗畬鏁寸粏鑳炲拰鑳屾櫙鐗╄川锛堝洜涓虹粏鑳炲拰鍏朵粬鐗╄川鐩告瘮锛岀湡姝ｇ粏鑳炰腑浼氭湁鏇村鐨刄MI锛岃€屽叾浠栫墿璐ㄥ彲鑳芥病鏈夋垨鑰呯敱浜庝竴浜涙薄鏌撹兘鎹曡幏鍒板皯閲忕殑杞綍鏈紝鎵€浠ヤ細鍑虹幇绗竴涓笅闄嶇殑瓒嬪娍锛夛紱绗?涓笅闄嶇殑瓒嬪娍锛氬尯鍒嗙粏鑳炵殑璐ㄩ噺锛屾崟鑾风巼浣庢垨缁嗚優鐮寸锛堣繖绫荤粏鑳炰腑鍩哄洜鏁颁細寰堝皯锛屽鑷碪MI鏁颁篃灏戯級锛岃€屾甯哥殑缁嗚優涓璘MI澶氫笖鍒嗗竷姣旇緝鎺ヨ繎锛屾墍浠ヨ川閲忓ソ鍜屼笉濂界殑缁嗚優鍦║MI涓婁篃浼氬瓨鍦ㄥ緢澶х殑宸紓锛岄殢鍚庡氨鍑虹幇浜嗙2涓笅闄嶈秼鍔裤€?/p>

褰撴暟鎹嚭鐜颁簡杩欎袱涓笅闄嶈秼鍔匡紝涓斿湪钃濊壊鍖哄煙鐨勭嚎鏉℃瘮杈冨钩绋虫椂锛屼篃鑳借鏄庢垜浠殑鏁版嵁璐ㄩ噺濂絶

4. 娴嬪簭缁撴灉缁熻

缁х画寰€涓嬭蛋锛屼笅涓€閮ㄥ垎鏄祴搴忕殑淇℃伅锛屽寘鎷€荤殑reads鏁扮洰浠ュ強涓€浜涜川鎺х殑鎸囨爣锛屼竴鑸儏鍐典笅Q30>90%琛ㄦ槑璐ㄩ噺鏄浉褰撲笉閿欑殑銆?/p>

褰撴垜浠湅鏁版嵁鐨勬椂鍊欙紝濡傛灉閬囧埌涓€浜涙寚鏍囦笉澶槑鐧芥槸浠€涔堟剰鎬濓紝澶у鍙互鐐瑰嚮宸︿笂瑙掔殑锛燂紝闅忓悗浼氬垪鍑轰笅鍒楁寚鏍囩殑瑙ｉ噴銆?/p>

5. 姣斿缁撴灉缁熻

鎶ュ憡涓櫎浜嗕細缁欏嚭娴嬪簭淇℃伅浠ュ锛屼篃浼氱粰鍑轰笌鍩哄洜缁勭殑姣斿淇℃伅锛屼富瑕佸寘鎷珿enome銆両ntergenic銆両ntronic銆?Exonic銆乀ranscriptome銆丄ntisense to Gene锛堣涓嬪浘锛夈€?/p>

铏界劧娴嬪簭鍜屾瘮瀵圭粨鏋?/strong>閮芥槸涓€浜涘父瑙勭殑璐ㄦ帶淇℃伅锛屽綋鎴戜滑鏁版嵁涓€鍒囨甯哥殑鏃跺€欙紝鐪嬭繖浜涙寚鏍囧彲鑳芥病鏈夐偅涔堥噸瑕侊紝浣嗘槸涓€鏃︽垜浠殑鏁版嵁姣旇緝濂囨€殑鏃跺€欙紝渚嬪鍙戠幇妫€娴嬪埌鐨勭粏鑳炴暟杩樿锛屼絾鏄熀鍥犳暟鐗瑰埆灏戯紝杩欎釜鏃跺€欐祴搴忓拰姣斿缁撴灉灏辩浉褰撻噸瑕佷簡锛佷箣鍓嶉亣鍒颁竴涓暟鎹氨鏄娴嬪埌鐨勫熀鍥犳暟鐗瑰埆灏戯紝鐒跺悗鑱氱被鐨勬椂鍊欏氨缁撴灉寰堝樊锛屽悗鏉ュ氨杩斿洖鍘荤湅杩欎簺璐ㄦ帶淇℃伅锛屾儕濂囩殑鍙戠幇寰堝reads閮芥槸姣斿鍒颁簡鍩哄洜闂村尯锛?/p>

鎵€浠ユ祴搴忕殑reads鏍规湰灏辨病鏈夎惤鍦ㄥ熀鍥犱笂锛屽鑷翠簡鏈€缁堟瘡涓粏鑳炴娴嬪埌鐨勫熀鍥犻潪甯稿皯锛岀劧鍚庡啀鍘荤户缁線涓嬫壘鍘熷洜銆?/p>

銆傛墍浠ュ憖锛岃繕鏄緱澶氱湅鏁版嵁锛屼粠閭ｄ互鍚庯紝鏁版嵁涓嬫潵閮戒細鍏堢湅鐪嬭繖浜涜川鎺т俊鎭槸鍚︽甯革紝鎵嶄細缁х画寰€鍚庡仛锛堣川鎺т篃鏄仛绉戠爺闈炲父閲嶈鐨勪竴姝ュ憖~锛?/p>

6. 鏍锋湰淇℃伅

鏈€鍚庝竴閮ㄥ垎灏辨槸鏍锋湰淇℃伅鍟︼紙濡備笅鍥撅級~

杩欎竴閮ㄥ垎灏辨槸鍦ㄨ繍琛孋ell Ranger鏃跺€欑殑鍙傛暟淇℃伅锛屼緥濡傛牱鏈悕銆丆hemistry锛堣繍琛孋ell Ranger鏃跺€欐垜浠病鏈夎缃繖涓弬鏁帮紝閭ｄ箞灏遍粯璁ら€夋嫨auto锛氳嚜鍔ㄩ厤缃紝鍦ㄦ姤鍛婁腑浼氱粰鍑哄叿浣撶殑绫诲瀷锛岃繖涓氨鏄?' V3鐗堟湰锛夈€丷eference浠ュ強Reference璺緞绛夌瓑銆傝繖浜涗俊鎭殑缁欏嚭鏂逛究鍚庨潰鏌ユ壘淇℃伅銆?/p>

05 Analysis

浠嬬粛瀹?Summary涔嬪悗锛屼笅闈㈠氨鏄疉nalysis.

1. 鍒嗙兢缁撴灉

宸﹀浘锛氬湪TNSE涓槧灏勬瘡涓粏鑳濽MI鐨勫€硷紱鍙冲浘锛歍SNE涓垎缇ょ殑鎯呭喌銆?/p>

Cell Ranger鍋氬畬瀹氶噺涔嬪悗鍛紝浼氶粯璁ゆ嬁宸叉湁鐨勭粨鏋滆窇涓€涓嬪熀鏈殑鍒嗙兢锛屾墍浠ュ湪鐪嬫姤鍛婄殑鏃跺€欐垜浠篃鍙互鐪嬩竴涓嬭繖閲岀殑鍒嗙兢缁撴灉锛屽績閲屽ぇ姒傛湁涓暟~

2. 鍩哄洜宸紓琛ㄨ揪鍒嗘瀽

Cell Ranger闄や簡鍋氫簡鍒嗙兢浠ュ锛岃繕鎵句簡姣忎釜缇ゅ樊寮傝〃杈剧殑鍩哄洜锛岀被浼间簬Seurat涓殑 "FindAllMarkers"銆?/p>

杩欓噷姣旇緝濂界殑鏄紝涓婇潰Graph-based濡傛灉閫夋嫨K=2锛岄偅涔堣繖閲屽樊寮傚熀鍥犲垪琛ㄤ篃浼氶殢涔嬪彉鍔ㄣ€傛墍浠ュ鏋滆寰桟ell Ranger鐨勫垎缇ょ粨鏋滃凡缁忓緢绗﹀悎鑷繁鐨勯鏈熶簡锛屽畬鍏ㄥ彲浠ュ氨鐢ㄨ繖涓粨鏋滀簡锛岃€屼笖杩樺彲浠ヨ嚜宸遍€夋嫨鍒嗙兢鐨勪釜鏁帮紙鐩存帴缃戦〉鎸戦€夛紝浜烘€у寲鍛€锛?/p>

3. 楗卞拰搴﹁瘎浼?/strong>

瀵?reads 鎶芥牱锛岃绠椾笉鍚屾娊鏍锋潯浠朵笅妫€娴嬪埌鐨勮浆褰曟湰鏁伴噺鍗犳娴嬪埌鐨勬墍鏈夎浆褰曟湰鐨勬瘮渚嬶紙娴嬪簭楗卞拰搴?锛屽涓嬪浘锛?/p>

鏇茬嚎鏈鎺ヨ繎骞虫粦鐘舵€佽鏄庢祴搴忚揪鍒伴ケ鍜岋紝鍥犱负缁х画澧炲姞娴嬪簭閲忥紝妫€娴嬪埌鐨勮浆褰曟湰涔熶笉浼氭湁鐗瑰埆澶х殑鍙樺寲

瀵?reads 鎶芥牱锛岃绠椾笉鍚屾娊鏍锋潯浠朵笅妫€娴嬪熀鍥犳暟鐩殑鍒嗗竷锛屽涓嬪浘锛?/p>

鍚屾牱鍦帮紝鏇茬嚎鏈鎺ヨ繎骞虫粦鐘舵€佽鏄庢祴搴忚揪鍒伴ケ鍜岋紝鍥犱负缁х画澧炲姞娴嬪簭閲忥紝姣忎釜缁嗚優妫€娴嬪埌鐨勫熀鍥犳暟涔熶笉浼氭湁鐗瑰埆澶х殑鍙樺寲

涓嬫父 barcodes.tsv.gz/features.tsv.gz/matrix.mtx.gz

cellranger count杈撳嚭缁撴灉涓殑outs.鏂囦欢澶规湁鍑犱釜鏄潪甯搁噸瑕佺殑淇℃伅锛屾垜浠粖澶╁彧鍏虫敞浜?strong>filtered_feature_bc_matrix鏂囦欢澶逛笅鐨勫唴瀹瑰拰possorted_genome_bam.bam鏂囦欢銆?/p>

涓€鑸潵璇达紝鎴戜滑涓嬫父鐨?strong>Seurat鍒嗘瀽鐨勮緭鍏ユ枃浠朵細閫夋嫨filtered_feature_bc_matrix涓殑鏂囦欢锛岃€屼笉閫夋嫨raw_feature_bc_matrix涓嬬殑鏂囦欢锛屽墠鑰呮槸缁忚繃杩囨护鐨勶紝鍘绘帀浜嗕綆璐ㄩ噺鐨勪俊鎭€傝繘鍏?strong>filtered_feature_bc_matrix鏂囦欢澶逛細鍙戠幇瀹冧笅闈㈠寘鍚?涓枃浠讹細鍒嗗埆鏄?strong>barcodes.tsv.gz銆?strong>features.tsv.gz鍜?strong>matrix.mtx.gz銆?/p>

barcodes.tsv.gz

AAACCCAAGAGATGCC-1
AAACCCAAGGTCGTAG-1
AAACCCACATCAGTCA-1
AAACCCAGTTTCCCAC-1
AAACCCATCCAAACCA-1
AAACCCATCCCTCTAG-1
AAACGAAAGCTGGTGA-1
AAACGAACAGACACAG-1
AAACGAAGTGAGATAT-1

杩欎釜鏂囦欢褰撲腑璁拌浇浜嗘瘡涓粏鑳炵殑barcode淇℃伅銆?/p>

features.tsv.gz

ENSMUSG00000051951      Xkr4    Gene Expression
ENSMUSG00000089699      Gm1992  Gene Expression
ENSMUSG00000102331      Gm19938 Gene Expression
ENSMUSG00000102343      Gm37381 Gene Expression
ENSMUSG00000025900      Rp1     Gene Expression
ENSMUSG00000025902      Sox17   Gene Expression
ENSMUSG00000104238      Gm37587 Gene Expression
ENSMUSG00000104328      Gm37323 Gene Expression

杩欎釜鏂囦欢璁拌浇浜嗗皬榧犲熀鍥犳敞閲婃枃浠朵腑鍖呭惈鐨勫熀鍥爄d涓巗ymbol淇℃伅锛屾敞鎰忥紝杩欎釜鏂囦欢鐨勬潵婧愭槸灏忛紶鍩哄洜缁勭殑娉ㄩ噴鏂囦欢銆?/p>

matrix.mtx.gz

%%MatrixMarket matrix coordinate integer general
%metadata_json: {"software_version": "cellranger-6.0.1", "format_version": 2}
32285 5741 11436472
1 1 4
2 1 1
22 1 1
24 1 8
31 1 1
41 1 1
43 1 1

杩欎釜鏂囦欢涓讳綋閮ㄥ垎鍖呭惈涓夊垪锛岀涓€鍒椾负鍩哄洜锛屽嵆杩欎釜鍩哄洜鍦ㄥ墠闈?strong>features.tsv.gz涓殑浣嶇疆锛涚浜屽垪涓虹粏鑳烇紝鍗宠繖涓粏鑳炲搴斾簬barcodes.tsv.gz涓殑barcodes淇℃伅锛涙渶鍚庝竴鍒椾唬琛ㄥ湪杩欎釜缁嗚優涓娴嬪埌鐨勮繖涓熀鍥犵殑reads鏁般€備妇涓緥瀛愭潵璇达細
渚嬪绗竴琛岋細1 1 4锛屽氨琛ㄧずbarcode涓篈AACCCAAGAGATGCC-1鐨勭粏鑳炰腑妫€娴嬪埌鐨刋kr4鍩哄洜鐨剅eads鏁颁负4銆?br> 缁嗗績鐨勬湅鍙嬩細鍙戠幇鍦ㄥ墠闈㈣繕鏈変竴琛岋細32285 5741 11436472 锛岃繖涓€琛屽疄闄呬笂灏辨槸涓€涓眹鎬讳俊鎭紝渚嬪鏈?2285涓熀鍥狅紝5741涓粏鑳烇紝11436472涓潪闆舵暟鍊笺€傝€屾渶鍓嶉潰涓嶈繃鏄寚鏄庤蒋浠剁殑鐩稿叧淇℃伅缃簡銆?/p>

鎬濊€?/h2>

瀹為檯涓婂湪鎴戜滑杩涜鏁版嵁鍒嗘瀽鏃讹紝閮借寰楄繖3涓枃浠朵竴涓笉鍙皯锛屼絾瀹為檯涓婄湡鐨勬槸杩欐牱鍚楋紵

• features.tsv.gz

鍓嶉潰宸茬粡璇村埌锛岃繖涓枃浠跺疄闄呬笂鏄潵婧愪簬灏忛紶鍩哄洜缁勭殑娉ㄩ噴鏂囦欢锛屾墍浠ョ悊璁轰笂鍙浣犲湪浣跨敤cellranger count鏃剁敤鐨勫熀鍥犵粍娉ㄩ噴鏂囦欢鏄竴鏍风殑锛岃繖涓枃浠舵槸涓嶄細鍙樼殑锛屼綘鍙互杩涘叆Cell Ranger鎺ㄨ崘鐨勫弬鑰冨熀鍥犵粍鐪嬫槸鍚︽槸杩欐牱銆?/p>

cd cellranger/reference/refdata-gex-mm10-2020-A/genes
#杩欎釜鏂囦欢澶逛笅闈綘浼氱湅鍒颁竴涓皬榧犲熀鍥犵粍鐨刧tf娉ㄩ噴鏂囦欢锛屽悕绉板簲璇ヤ负genes.gtf
cat genes.gtf | awk '=="gene_name"{print"\t"}' | less -S
#鐪嬬湅杩欐牱鎻愬彇鐨勫熀鍥爄d鍜宯ame鏄惁鍜宖eatures.tsv.gz涓€鏍?
"ENSMUSG00000051951";   "Xkr4";
"ENSMUSG00000089699";   "Gm1992";
"ENSMUSG00000102331";   "Gm19938";
"ENSMUSG00000102343";   "Gm37381";
"ENSMUSG00000025900";   "Rp1";
"ENSMUSG00000025902";   "Sox17";
"ENSMUSG00000104238";   "Gm37587";

浣犱細鍙戠幇锛岄『搴忓拰鍐呭绔熺劧鍜?strong>features.tsv.gz涓€鏍风殑锛屾墍浠ョ湅璧锋潵浼间箮features.tsv.gz涔熶笉鏄偅涔堜笉鍙垨缂猴紝鍜变篃鍙互鑷繁鍋氾紝鎴栬€呰鍙互閫氱敤銆?/p>

• matrix.mtx.gz

杩欎釜鏂囦欢锛屾鏃犵枒闂紝鏄繀涓嶅彲灏戠殑锛屽彲浠ヨ鑺遍偅涔堝閽卞仛涓猻ingle cell RNA sequencing灏辨槸涓轰簡杩欎釜鏂囦欢銆傘€?/p>

• barcodes.tsv.gz

鍏夊惉杩欎釜鏂囦欢鐨勫唴瀹癸紝鎰熻杩欎釜鏂囦欢寰堥噸瑕侊紝鍍忔煇涓湴鍖哄眳姘戠殑鍚嶅崟涓€鏍凤紝涓簡宀備笉楹荤儲澶т簡锛熶絾瀹為檯涓婁粩缁嗘兂鎯筹紝瀹冪湡鐨勯噸瑕佸埌鎴戜滑涓嶈兘涓㈠悧锛?br> 鎴戜滑璇达紝matrix.mtx.gz閲岄潰瀹為檯涓婂凡缁忓寘鍚簡鍗曚釜缁嗚優銆佸崟涓熀鍥犵殑琛ㄨ揪淇℃伅浜嗭紝杩欐槸cellranger count宸茬粡杩旂粰鎴戜滑鐨勪俊鎭紝涓句釜褰㈣薄鐨勪緥瀛愶紝灏忓瀛愬湪鍑虹敓鏃讹紝褰撳湴鎴风睄閮ㄩ棬璁板綍浜嗚繖涓皬瀛╃殑鎬у埆淇℃伅锛屽綋鐒惰繕鏈変粬鐨勫悕瀛椼€備絾鏄竴骞村悗锛岃繖涓皬鏈嬪弸鏀瑰悕瀛椾簡锛屼絾鏄粬鐨勬€у埆鍙樹簡鍚楋紵骞舵病鏈夛紒鎵€浠ュ疄闄呬笂杩欎釜barcodes.tsv.gz鏂囦欢濡傛灉鎴戜滑鏀逛簡锛屽彧涓嶈繃鏄粰姣忎釜缁嗚優鏂拌捣浜嗕竴涓悕瀛楋紝鏈韩骞朵笉浼氶€犳垚缁嗚優RNA淇℃伅鐨勫彉鍖栧拰娣蜂贡銆?/strong>
璇村埌杩欓噷锛屼笉寰椾笉鎻愬埌possorted_genome_bam.bam鏂囦欢锛岃繖涓枃浠堕噷闈㈠疄闄呬笂鍖呭惈浜嗘瘡涓粏鑳炵殑barcode淇℃伅锛屽氨鍦ㄥ叾涓互CB寮€澶寸殑閭ｄ釜瀛楁閲屻€?/p>

samtools view possorted_genome_bam.bam | less -S
#閮ㄥ垎淇℃伅濡備笅
CB:Z:ATTCTTGTCTCCTGTG-1
CB:Z:GTGCTGGTCACTCGAA-1
CB:Z:GCATGATAGCCGGATA-1
CB:Z:GCACGTGGTTGCCTAA-1

浣犲彲浠ユ妸杩欓儴鍒嗕俊鎭彁鍙栧嚭鏉ワ紝閲嶅鍐呭鍚堝苟锛岀劧鍚庝互浠绘剰椤哄簭浣滀负barcodes.tsv.gz灏卞彲浠ヨ繘琛孲eurat鍒嗘瀽浜嗐€傚摝瀵逛簡锛屽緱鏌愪綅澶т浆鎸囩偣锛宑ellranger count杈撳嚭鐨?strong>barcodes.tsv.gz鏄寜瀛楁瘝琛ㄩ『搴忕殑锛屾墍浠ワ紙璋佺煡閬撳畠鏄笉鏄渶鍚庨殢鎰忕敤瀛楁瘝琛ㄩ『搴忚緭鍑虹殑鍛紵锛?/p>

闇€浜嗚В

灏哛eads杞负 Count Matrix銆?/p>

1. 鍙傝€冨熀鍥犵粍鍙婃敞閲?/h3>

鐩墠锛屽ぇ澶氭暟scRNA-seq鏄娇鐢ㄤ汉绫绘垨灏忛紶鐨勭粍缁囥€佸櫒瀹樻垨缁嗚優鍩瑰吇鐗╄繘琛岀殑銆傚父鐢ㄧ殑灏辨槸UCSC锛坔g19銆乭g38銆乵m10绛夛級锛屽拰GRC锛圙RCh37銆丟RCh38銆丟RCm38锛夈€備簩鑰呭湪涓昏鏌撹壊浣撲笂鏄绛夌殑锛堝hg38鐨刢hr1 = GRCh38鐨刢hr1锛夛紝浣嗗湪涓€浜涘皬鐨勪綅鐐逛笂浼氭湁缁嗗井宸紓銆?/p>

鍩哄洜缁勬敞閲婅繃绋嬪寘鎷畾涔夊熀鍥犵粍鐨勮浆褰曞尯鍩燂紝鏄庣‘exon鍜宨ntron锛屽皢鍏跺垎鎴恜rotein coding, non-coding绛夈€備妇涓牀瀛愶紝鍋囪鎴戜滑鏈変竴涓熀鍥狅紝鍖呭惈5涓浆褰曟湰缁勬垚鐨勫熀鍥犮€傚叾涓?涓紪鐮侊紙绾㈣壊锛夊拰2涓潪缂栫爜锛堣摑鑹诧級銆?/p>

鍦ㄥ疄闄呮搷浣滀腑锛屾垜浠€氬父鍙互涓嬭浇GTF鎴朑FF3鏍煎紡鐨勬枃浠惰繘琛屾敞閲娿€傛瘡涓熀鍥犻兘鍚湁涓€涓狪D锛岃€岃繖涓狪D鏄敮涓€鐨勩€?/p>

Note! 杩欓噷涔熸彁閱掑ぇ瀹跺湪瀹為檯鎿嶄綔涓紝灏介噺浣跨敤ID杩涜鍒嗘瀽鎿嶄綔锛岃€屼笉瑕佷娇鐢╯ymbol锛屽綋鐒跺湪灞曠ず缁撴灉鐨勬椂鍊欎綘闇€瑕佽浆鎹㈠洖symbol鏂逛究闃呰銆?/p>

鎴戜滑甯哥敤鐨勪汉绫诲拰灏忛紶鍩哄洜缁勬敞閲婂寘鎷琑efSeq, ENSEMBL鍜孏ENCODE锛屽疄闄呭簲鐢ㄤ腑閫夋嫨鏈€鏂扮殑鐗堟湰灏卞彲浠ヤ簡锛屼細鏈夋洿澶氱殑宸茬煡鍩哄洜銆?/p>

2. Full-length scRNA-seq鐨勫鐞?/h3>

澶勭悊鏂规硶涓巄ulk RNA-seq绫讳技銆?/p>

Full length scRNA-seq鐨剅aw data鐨勫鐞嗛€氬父鍒嗕袱姝ヨ繘琛岋細姣斿(read alignment)鍜岃鏁?(read counting)銆傚父鐢ㄨ蒋浠讹細STAR鍜宧isat2銆俷ormalization鏂规硶锛氭帹鑽愪娇鐢═PM銆?/p>

Droplet-based scRNA-seq鐨勬瘮瀵瑰拰瀹氶噺
1锔忊儯 棣栧厛鎴戜滑瑕佹悶娓呮scRNA-seq閮芥湁鍝簺浜х墿銆?/p>

• cDNA鐗囨 (璇嗗埆杞綍鏈?锛?/li>
• Cell barcode (CB锛岃瘑鍒粏鑳?锛?/li>
• Unique Molecular Identifier (UMI锛屽噺灏廝CR鎵╁甯︽潵鐨刡ias)銆?/li>

2锔忊儯 鍏稿瀷鐨剆cRNA-seq鐨剋orkflow鍖呮嫭浠ヤ笅鍑犱釜姝ラ锛氿煈?/p>

• 灏哻DNAmapping鍒皉eference涓婏紱
• 璁＄畻鍩哄洜reads锛?/li>
• 璁＄畻缁嗚優reads锛堢敤鍒癱ell barcode锛夛紱
• 璁＄畻鐨凴NA鏁伴噺锛圲MI鍘婚噸锛夈€?/li>

3. 鍏蜂綋姝ラ

Read Mapping
澶勭悊10x Genomics Chromium scRNAseq鏁版嵁锛屾垜浠€氬父瑕佺敤鍒癈ell Ranger銆傝繖閲屽彧浠嬬粛涓€涓嬪鏄惧瓙(exon)鐨勫畾涔夛紝鍗硆eads姣斿鍒板鏄惧瓙鐨?50% 浠ヤ笂锛屽氨鍙互瀹氫箟涓哄鏄惧瓙銆?/p>

Cell Ranger Reference
鍦ㄩ€夋嫨Reference鐨勬椂鍊欙紝澶у鍙互鎸変互涓媡able杩涜閫夋嫨銆傪煈?/p>

Cell Ranger Reference   Species Assembly/Annotation Genes before filtering  Genes after filtering
2020-A  human   GRCh38/GENCODE v32  60668   36601
2020-A  mouse   mm10/GENCODE vM23   55421   32285
3.0.0   human   GRCh38/Ensembl 93   58395   33538
3.0.0   human   hg19/Ensembl 87 57905   32738
3.0.0   mouse   mm10/Ensembl 93 54232   31053
2.1.0   mouse   mm10/Ensembl 84 47729   28692
1.2.0   human   GRCh38/Ensembl 84   60675   33694
1.2.0   human   hg19/Ensembl 82 57905   32738
1.2.0   mouse   mm10/Ensembl 84 47729   27998

UMI璁℃暟
杩欓噷鎴戜滑娉ㄦ剰涓€涓嬩袱鐐癸細

• 濡傛灉涓ょ粍鎴栨洿澶氱殑reads鍏锋湁鐩稿悓鐨刡arcode鍜孶MI锛屼絾鍩哄洜娉ㄩ噴鍗翠笉鐩稿悓锛岄偅涔坮eads鏈€澶氱殑鍩哄洜娉ㄩ噴鐢ㄤ簬UMI璁℃暟锛岃垗寮冨叾浠栫粍銆?/p>

• 鎴戜滑鍐嶆瀬绔竴鐐癸紝濡傛灉涓ょ粍reads涓€鏍风殑璇濓紝杩欎釜鏃跺€欐垜浠彲鑳介渶瑕佽垗寮冩墍鏈夌粍锛屽洜涓鸿繖涓椂鍊欏熀鍥犳敞閲婂凡缁忎笉鍑嗙‘浜嗐€?/p>

缁嗚優杩囨护
1锔忊儯 鏈粡杩囨护鐨剅aw data, feature-barcode matrix浼氬寘鍚緢澶氱┖鐨?droplets锛屽湪鐭╅樀涓苟涓嶆槸0锛屽洜涓轰細鏈夋潵鑷牬纰庣粏鑳炵殑RNA銆傛墍浠ワ紝杩欑鏁版嵁鎴戜滑闇€瑕佽繘琛岃繃婊わ紝鑰屽悗鍐嶈繘琛屽垎鏋愩€?/p>

2锔忊儯 鎴戜滑閫氬父闇€瑕佷娇鐢–ell Ranger 2.2鍜孋ell Ranger 3.0杩涜杩囨护銆?/p>

3锔忊儯 涓句釜鏍楀瓙

鑲跨槫鏍锋湰閫氬父鍖呭惈澶у瀷鑲跨槫缁嗚優涓庡皯閲忕殑鑲跨槫娴告鼎娣嬪反缁嗚優锛圱IL锛夛紝濡傛灉浣犲TIL鐗瑰埆鎰熷叴瓒ｏ紝閭ｈ繖涓椂鍊欏彲鑳藉氨瑕佺敤鍒癊mptyDrops鐨勬柟娉曟潵杩涜杩囨护浜嗐€?/p>

鍏朵粬鏂规硶
褰撲綘浣跨敤Cell Ranger鏃讹紝浣犲彲鑳戒細瑙夊緱瀹冧笉澶熷揩锛岃繖閲屾垜浠粙缁嶄袱涓€熷害蹇€佸噯纭害楂樼殑鏂规硶锛孲TARsolo鍜孉levin锛岃繖閲屼笉鍋氬叿浣撲粙缁嶄簡锛屾帹鑽愬ぇ瀹堕€夋嫨STARsolo銆?/p>

鍙傝€冿細
https://www.jianshu.com/p/3f01016b5302
https://mp.weixin.qq.com/s/iB1o4BhCElU5IExOtXbtmw
https://mp.weixin.qq.com/s/9QA20YQEe1PPkDarB964HQ